AXL kinase inhibitors and use of the same

ABSTRACT

Tartrate salts of the compound of structure (I),crystalline forms thereof, and therapeutic applications thereof for treating solid tumors (e.g., advanced solid tumor) or hematopoietic cancers. Also provided herein are methods for synthesizing the tartrate salts and the crystalline forms thereof.

CROSS-REFERENCE

This application is a Divisional application of U.S. application Ser.No. 16/376,452, filed Apr. 5, 2019, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application, U.S. Ser. No.62/813,705, filed Mar. 4, 2019, U.S. Provisional Application, U.S. Ser.No. 62/778,856, filed Dec. 12, 2018, U.S. Provisional Application, U.S.Ser. No. 62/767,475, filed Nov. 14, 2018, U.S. Provisional Application,U.S. Ser. No. 62/760,882, filed Nov. 13, 2018, U.S. ProvisionalApplication, U.S. Ser. No. 62/698,638, filed Jul. 16, 2018, U.S.Provisional Application, U.S. Ser. No. 62/695,609, filed Jul. 9, 2018,U.S. Provisional Application, U.S. Ser. No. 62/688,161, filed Jun. 21,2018, U.S. Provisional Application, U.S. Ser. No. 62/678,980, filed May31, 2018, U.S. Provisional Application, U.S. Ser. No. 62/663,146, filedApr. 26, 2018, and U.S. Provisional Application, U.S. Ser. No.62/653,394, filed Apr. 5, 2018, the entire contents of each of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

AXL is a cell surface receptor tyrosine kinase of the TAM family. TheAXL receptor binds growth factors like vitamin K-dependent proteingrowth-arrest-specific gene 6 (GAS6) and transduces signals from theextracellular matrix into the cytoplasm. It is reported that AXL is aninhibitor of the innate immune response and may play a role in multiplecellular processes relating to cell growth and development.

AXL is found to be involved in various aspects of tumor growth,including cancer cell proliferation, invasiveness and migration, as wellas sternness, angiogenesis, and immune modulation. As such, AXL becomesa promising cancer treatment target.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a tartrate salt of thecompound of structure (I):

In some embodiments, the tartrate salt disclosed herein may have a molarratio of tartaric acid to the compound of structure (I) of about 1:1 toabout 2:1. For example, the tartrate salt may have a molar ratio oftartaric acid to the compound of structure (I) of about 2:1. Any of thetartrate salts disclosed herein may be a salt of L-(+)-tartaric acid.

In another aspected, the present disclosure provides a crystalline formof any of the tartrate salts disclosed herein. In some embodiments, thecrystalline form is crystalline Form A, having a molar ratio of tartaricacid to the compound of structure (I) of about 2:1. In some examples,Form A can be in substantially pure form. In some embodiments, thecrystalline form comprises Form A. In some embodiments, the crystallineform consists essentially of Form A.

Any of the crystalline forms disclosed herein may be characterized by anx-ray powder diffraction (XRPD) pattern comprising peaks, in units2-theta, at 11.2±0.2, 17.1±0.2, and 19.9±0.2. Optionally, thecrystalline form may further comprise a peak, in units 2-theta, at15.4±0.2. Alternatively or in addition, the crystalline form may furthercomprise a peak, in units 2-theta, at 7.0±0.2. In some examples, thecrystalline form as disclosed herein can be characterized by an XRPDpattern comprising three or more peaks, in units of 2-theta, selectedfrom 7.0±0.2, 11.2±0.2, 15.4±0.2, 16.3±0.2, 17.1±0.2, 19.9±0.2,21.6±0.2, and 25.5±0.2. In other examples, the XRPD pattern may comprise4, 5, 6, 7, or 8 peaks, in units of 2-theta, selected from 7.0±0.2,11.2±0.2, 15.4±0.2, 16.3±0.2, 17.1±0.2, 19.9±0.2, 21.6±0.2, and25.5±0.2. In one specific example, the XRPD pattern is substantiallyidentical to the XRPD pattern shown in FIG. 61.

Alternatively or in addition, any of the crystalline forms disclosedherein may be characterized by a differential scanning calorimetry (DSC)thermogram comprising an endotherm peak in units ° C. of about185.0-194.0. In some embodiments, the endotherm peak has an onsettemperature of about 186.3° C. In some embodiments, the crystalline formas disclosed herein may be characterized by a DSC thermogram comprisingendotherm peaks in units ° C. at about 107.8, about 152.1, and about189.1. In one particular example, the crystalline form may have a DSCthermogram that is substantially identical to the thermogram shown inFIG. 64.

Further, any of the crystalline forms disclosed herein may becharacterized by a thermogravimetric analysis (TGA) thermogram showingweight loss of about 1.8% at 160° C. For example, the TGA thermogram issubstantially identical to the thermogram shown in FIG. 64.

Any of the crystalline forms disclosed herein may have an initial purityof at least 99% and a subsequent purity of at least 99% after beingstored for up to about 15 days at about 25° C.±2° C. at a relativehumidity of 60±5%. In some embodiments, the crystalline form may aninitial purity of at least 99% and a subsequent purity of at least 99%after being stored for up to about 15 days at about 40° C.±2° C. at arelative humidity of 75±5%.

Also within the scope of the present disclosure is a compositioncomprising any of the tartrate salts disclosed herein or any of thecrystalline forms also disclosed herein. In some embodiments, thecomposition comprises Form A in substantially pure form. In someembodiments, the composition comprises at least 90% Form A by weight. Insome embodiments, the composition consists essentially of crystallineForm A.

Any of the compositions disclosed herein may be a pharmaceuticalcomposition and further comprises a pharmaceutically acceptable carrieror diluent. In some embodiments, the pharmaceutical composition mayfurther comprises one or more additional therapeutic agents, forexample, hormone therapeutic agents, chemotherapeutic agents,immunotherapeutic agents, cell growth factors, or agents to inhibit cellgrowth factor receptor action. Any of the pharmaceutical compositionsdisclosed herein may be formulated for oral administration. For example,the pharmaceutical composition may be formulated as a capsule or tablet.

Further, the present disclosure provides a unit dose comprising any ofthe pharmaceutical compositions disclosed herein. The unit dose maycomprise about 1-100 mg of the tartrate salt. In some embodiments, theunit dose disclosed herein may comprise about 1 mg, 4 mg, 16 mg, 25 mg,50 mg, 75 mg, or 100 mg of the tartrate salt. In some embodiments, theunit dose can be formulated in a gelatin hard capsule for oraladministration.

In another aspect, the present disclosure provides a method of treatinga cancer, the method comprising administering to a subject (e.g., ahuman) in need thereof a therapeutically effective amount of any of thetartrate salts disclosed herein, any of the crystalline forms disclosedherein, any of the pharmaceutical compositions comprising such, or anyof the unit doses also disclosed herein.

In some embodiments, the method disclosed herein comprises administeringto the subject a therapeutically effective amount of a tartrate salthaving a molar ratio of tartaric acid to the compound of structure (I)of about 2:1. In some examples, the method comprises administering tothe subject a therapeutically effective amount of the crystalline FormA, which may be in substantially pure form.

In a further aspect, provided herein is a method of treating cancer in asubject comprising administering to the subject a compound of structure(I):

or a pharmaceutically acceptable salt or crystalline form thereof. Thecompound, or pharmaceutically acceptable salt, or crystalline formthereof, can be administered to the subject orally once daily at a dosefrom about 1.5 mg/m² to about 65 mg/m², or at a dose of about 20 mg toabout 100 mg. The subject may have any of the tumor (e.g., advancedsolid tumor) or hematopoietic cancer as disclosed herein. In someembodiments, the subject may have undergone one or more prior cancertherapy, e.g., those disclosed herein.

In some embodiments, the compound of structure (I), or pharmaceuticallyacceptable salt, or crystalline form thereof, can be administered orallyat a dose of about 1.0, about 1.5, about 3.0, about 6.0, about 9.0,about 12.0, about 16.0, about 21.0, about 28.0, about 37.0, about 49.0,or about 65.0 mg/m². For example, the compound of structure (I), orpharmaceutically acceptable salt, or crystalline form thereof, can beadministered to the subject as a single anti-cancer agent at a dailydose of about 25 mg, about 33 mg, about 45 mg, about 50 mg, about 58 mg,about 75 mg, or about 100 mg. In specific examples, the compound ofstructure (I), or pharmaceutically acceptable salt, or crystalline formthereof, is administered orally to the subject concurrently with asecond anti-cancer agent and the daily dose of the compound of structure(I), or pharmaceutically acceptable salt, or crystalline form thereof isabout 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg,about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, orabout 100 mg.

In any of the methods disclosed herein, the cancer can be a solid tumoror a hematological cancer. Examplary solid tumors include, but are notlimited to, a tumor of the bones, digestive organs, reproductive organs,head, neck, lung, heart, skin, nervous system, endocrine system,neuroendocrine system, urinary system, soft tissue, or brain. Examplaryhematopoietic cancers include, but are not limited to, multiple myeloma,myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), acutelymphoblastic leukemia (ALL), acute lymphocytic leukemia, chroniclymphogenous leukemia, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), mantle cell lymphoma, diffuse large B-celllymphoma, follicular lymphoma, or non-Hodgkin's lymphoma.

In some embodiments, the cancer is an advanced solid tumor and/or hasshown disease progress after receiving one or more prior cancer therapy.For example, the solid tumor can be a resistant, recurrent, refractory,or relapsed solid tumor. In some examples, the one or more prior cancertherapy comprises a hormone therapeutic agent, a chemotherapeutic agent(e.g., an alkylating agent, a platinum-based agent, an antimetabolite,an anti-cancer antibiotic, or a plant-derived anti-cancer agent), animmunotherapeutic agent, or a cell surface receptor inhibitor. Exemplarychemotherapeutic agents include, but are not limited to, carboplatin,cisplatin, miboplatin, nedaplatin, and oxaliplatin.

In some embodiments, the subject has undergone one or more prior cancertherapy, which may comprise immunotherapy, chemotherapy, or acombination thereof.

In some embodiments, the subject has a solid tumor, which may benon-small cell lung cancer (NSCLC), colorectal carcinoma (CRC), ovariancancer, melanoma, breast carcinoma, neurodocrine carcinoma, prostateadenocarcinoma, cholangiocarcinoma, uterine carcinoma, and pancreaticcancer.

In some embodiments, the subject may have at least stable disease afterreceiving an immunotherapy. For example, the subject may have receivedup to two cycles of the immunotherapy.

In some embodiments, the subject may have EGFR⁺ NSCLC. In some examples,the subject may have demonstrated disease progression after receivingone or more chemotherapies comprising one or more tyrosine kinaseinhibitors (TKIs), which may comprise an EGFR TKI. In some examples, thesubject may have at least stable disease after taking up to two lines ofthe TKIs.

In some embodiments, the subject may have BRAF-, KRAS-, or NRAS-mutatedcolorectal carcinoma (CRC).

In some embodiments, the subject may have persistent or recurrentovarian cancer. For example, the subject may have refractory orresistant to a platinum-based agent, has undergone prior therapy, orboth. In some examples, the platinum-based agent is carboplatin,cisplatin, miboplatin, nedaplatin, or oxaliplatin.

In some embodiments, the subject may have BRAF-mutated melanoma. In someexamples, the subject may have progressed disease after animmunotherapy, a chemotherapy comprising one or more BRAF/MEKinhibitors, or a combination thereof.

In some embodiments, the subject may be resistant to an immunotherapyand wherein the method further comprising subjecting the subject to thesame immunotherapy. In some examples, the immunotherapy comprises ananti-PD-1 or an anti-PD-L1 agent.

In any of the methods disclosed herein (e.g., a method for treatingEGFR+ NSCLC), the subject may be resistant to a chemotherapy comprisinga TKI. Such a method may further comprise subjecting the subject to thesame chemotherapy.

In any of the methods disclosed herein, the subject is not takingsteroids at an amount equivalent to 15 mg/day of prednisone.Alternatively or in addition, the method may further comprisesadministering to the subject concomitant steroids. In some embodiments,the subject may be free of an anticancer therapy at least within themonth prior to the first dosing of the tartrate salt, free of CYP2C19metabolizers, and/or H2-receptor antagonists at least within 7 daysprior to the first dosing of the tartrate salt.

In any of the methods disclosed herein, the subject can be administeredorally the tartrate salt, the crystalline form thereof, or thepharmaceutical composition comprising such at a daily dose of about 1.0,1.5, 3.0, 6.0, 9.0, 12.0, 16.0, 21.0, 28.0, 37.0, 49.0, or 65.0 mg/m² ofthe tartrate salt. In some examples, the subject can be administeredorally the tartrate salt, the crystalline form thereof, or thepharmaceutical composition at a daily dose of the tartrate salt of about20-100 mg, optionally about 25-75 mg, for example, about 50 mg.

Any of the methods disclosed herein may comprise one or more a treatmentcycles, each consisting of about 28 days, and wherein in each treatmentcycle, the tartrate salt, the crystalline form thereof, or thepharmaceutical composition is administered to the subject daily for 21days, followed by a drug holiday period of seven days.

In other embodiments, the subject to be treated by any of the methodsdisclosed herein may have a hematological cancer, which can be chroniclymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In someembodiments, the subject has undergone one or more prior therapies forCLL or SLL. In some embodiments, the subject can be intolerant to theprior therapies or has progressive disease after the prior therapies. Insome examples, the prior therapies may comprise a B-cell receptorantagonist, a BCL-2 antagonist, or a combination thereof. In otherembodiments, the subject may not be concurrently treated by anothercancer therapy. In some examples, the subject may be administered orallythe tartrate salt, the crystalline form thereof, or the pharmaceuticalcomposition at a daily dose of about 20-100 mg, optionally about 25mg-100 mg of the tartrate salt, for example, about 25 mg, about 33 mg,about 45 mg, about 50 mg, about 58 mg, about 75 mg, or about 100 mg.

In some examples, the method for treating the hematopoietic cancer mayfurther comprise administering to the subject a BTK inhibitor, which maybe ibrutinib, acalabrutinib, zanubrutinib, and LOXO-305. In oneparticular example, the BTK inhibitor can be ibrutinib. In someexamples, the subject was subject to a prior treatment of the BTKinhibitor and has progressed on the BTK inhibitor treatment. In someexamples, the subject can be administered the tartrate salt, thecrystalline form thereof, or the pharmaceutical composition comprisingsuch at a daily dose of about 20 mg-100 mg (e.g., about 25-75 mg) of thetartrate salt, for example, about 20 mg, about 25 mg, about 33 mg, about45 mg, about 50 mg, about 58 mg, about 75 mg, or about 100 mg.

In some embodiments, any of the methods disclosed herein may compriseone or more treatment cycles, each consisting of about 28 days, andwherein in each cycle, the subject is administered the tartrate salt,the crystalline form thereof, or the pharmaceutical compositioncomprising such orally once daily for 28 days.

Any of the methods disclosed herein may further comprise (a) monitoringthe subject for tumor lysis syndrome (TLS); (b) administering to thesubject an antibiotic, an anti-viral agent, an anti-fungal agent, or acombination thereof, or a combination thereof.

In some embodiments, the subject may have an elevated level of solubleAXL, AXL expression and/or phosphorylation, growth arrest specific 6(GAS6), a mesenchymal transcription factor, or a combination thereof, asrelative to a reference level of soluble AXL, GAS6, or the mesenchymaltranscription factor. In some examples, the method may further comprise,prior to the administering step, identifying a subject having anelevated level of soluble AXL, AXL expression and/or phosphorylation,growth arrest specific 6 (GAS6), a mesenchymal transcription factor, ora combination thereof, as relative to a reference level of soluble AXL,GAS6, or the mesenchymal transcription factor. In some examples, theidentifying step can be performed by obtaining a peripheral blood sampleor a bone marrow sample of a candidate subject, and measuring the levelof soluble AXL, the level of GAS6, the level of the mesenchymaltranscription factor, or a combination thereof in the peripheral bloodsample or the bone marrow sample.

Any of the methods disclosed herein may further comprise, after theadministering step, examining the subject for one or more symptoms ofdiarrhea, nausea, vomiting, dysgeusia, anemia, and thrombocytopenia. Inaddition, the method may further comprise lowering the daily dose of thetartrate salt or terminating the treatment if the one or more symptomsare detected.

In some embodiments, the method disclosed herein may further compriseadministering an effective amount of one or more therapeutic agents tothe subject. The one or more therapeutic agents may comprise one or moretyrosine kinase inhibitors. In some examples, the one or more tyrosinekinase inhibitors may comprise an EGFR inhibitor. In other examples, theone or more therapeutic agents may comprises an immune checkpointinhibitor, for example, a PD-1 or PD-L1 inhibitor. Examples of PD-1inhibitors include, but are not limited to, pembrolizumab, nivolumab, ora combination thereof. Examples of PD-L1 inhibitors include, but are notlimited to, atezolizumab, avelumab, durvalumab, or a combinationthereof. Alternatively or in addition, the one or more therapeuticagents may comprise a CDK inhibitor, for example, a CDK9 inhibitor,which, in some instances, may be alvocidib, or a pharmaceuticallyacceptable salt or prodrug thereof. In some examples, the CDK9 inhibitoris a prodrug of alvocidib, which may have the following structure (II′):

or a pharmaceutically acceptable salt or zwitterionic form thereof.

In some examples, the one or more therapeutic agents comprise aplatinum-based chemotherapeutic agents.

Exemplary additional therapeutic agents to be used in combination withthe compound of structure (I), or a pharmaceutically acceptable saltthereof (e.g., any of the tartarate salts disclosed herein) may comprisecarboplatin, gemcitabine, bevacizumab, topotecan, rucaparib, olaparib,niraparib, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab,ipilimumab, or a combination thereof.

In yet another aspect, the present disclosure provides a methodcomprising administering an effective amount of a compound of structure(I):

or a pharmaceutically acceptable salt thereof, to a subject in needthereof, wherein the cancer is selected from the group consisting ofEGFR+ non-small cell lung cancer; BRAF-, KRAS-, or NRAS-mutatedcolorectal carcinoma; persistent or recurrent ovarian carcinoma;BRAF-mutated melanoma, inflammatory breast cancer, and triple negativebreast cancer. In some embodiments, the subject may have shown diseaseprogress after receiving one or more prior cancer therapy. For example,the cancer is a resistant, refractory, recurrent, or relapsed cancer. Insome embodiments, the one or more prior cancer therapy comprises ahormone therapeutic agent, a chemotherapeutic agent, animmunotherapeutic agent, or a cell surface receptor inhibitor.

In addition, the present disclosure provides a method of treating acancer, the method comprising administering an effective amount of acompound of structure (I):

or a pharmaceutically acceptable salt thereof, to a subject in needthereof, wherein the subject has an advanced solid cancer orhematopoietic cancer, wherein the subject shows disease progressionafter a chemotherapy, an immunotherapy, or a combination thereof. Thesubject to be treated by this method can have any of the solid tumor orhematopoietic cancer as disclosed herein. In some embodiments, thechemotherapy or immunotherapy may comprise a hormone therapeutic agent,a chemotherapeutic agent, an immunotherapeutic agent, or a cell surfacereceptor inhibitor, e.g., those disclosed herein. In some embodiments,the subject is resistant to an immunotherapy and wherein the methodfurther comprising subjecting the subject to the same immunotherapy. Insome embodiments, the subject is resistant to a chemotherapy comprisinga TKI, and wherein the method further comprising subjecting the subjectto the same chemotherapy.

Moreover, provided herein is a method for preparing a compound ofstructure (I):

or a pharmaceutically acceptable salt thereof. The method may comprisereacting a compound having the following structure:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   X′ is a leaving group;    -   Y is halo;    -   Z is halo or —NR¹(R²); and    -   R¹ and R² are, each independently, hydrogen or C1-C₈ alkyl, with        N-methylpiperazine, or a salt thereof, to obtain a compound        having the following structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the method may further comprise the step of:

-   -   reacting a compound having the following structure:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   Y is halo;    -   Z is halo or —NR¹(R²); and    -   R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl, with        an activating agent to obtain the compound having the structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the activating agent comprises a sulfonyl chloridefunctional group. In some embodiments, X′ is halo or sulfonate (e.g.,chloro). Alternatively or in addition, the activating agent is thionylchloride.

In some embodiments the method may further comprise the step:

reacting a compound having the following structure:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   Y is halo;    -   Z is halo or —NR¹(R²); and    -   R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl, with        a reducing agent to obtain the compound having the structure:

In some examples, the reducing agent is lithium aluminum hydride,diborane, sodium borohydride, borane, or combinations thereof. In oneparticular example, the reducing agent is borane.

Any of the methods disclosed herein may further comprise: reacting acompound having the following structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   X is a leaving group;    -   Y is halo;    -   Z is halo or —NR¹(R²); and    -   R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl, with        a compound having the following structure:

wherein P is H or a protecting group, to obtain a compound having thefollowing structure:

In some embodiments, X can be halo or sulfonate (e.g., chloro).Alternatively or in addition, P can be H; Y can be chloro; Z can be—NR¹(R²); R¹ can be C₁-C₈ alkyl (e.g., methyl); and/or R² can be C₁-C₈alkyl (e.g., methyl).

In yet another aspect, the present disclosure provides a method forpreparing a tartrate salt of the compound of structure (I):

The method may comprise admixing the compound of structure (I) withtartaric acid. In some examples, the tartaric acid can be L-(+)-tartaricacid. In some embodiments, the tartrate salt is of crystalline Form A,and the method comprises: (a) dissolving the compound of structure (I)in a solvent comprising anisole and ethanol to afford a first solution;(b) adding a solution of (L)-tartartic acid in ethanol to the firstsolution to afford a second solution; and (c) allowing the tartrate saltof structure (I) to crystallize from the second solution.

In some embodiments, the solvent of step (a) may comprise anisole andethanol in a ratio of about 2.5:1 (w/w) and/or has a temperature ofabout 70° C. In some examples, the molar ratio of (L)-tartratic acid instep (b) to the compound of structure (I) in step (a) is about 2:1and/or wherein the solution of tartaric acid is added to the firstsolution over the course of about an hour. In any of the methodsdisclosed herein, the second solution is cooled to about 20° C. and/oris stirred for about 5 hours.

Also within the scope of the present disclosure is a compound having thefollowing structure (III):

or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof,wherein:

-   -   Y is halo;    -   Z is halo or —NR¹(R²);    -   R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl;    -   R³ is halo or OR^(a);    -   R⁴ is hydrogen or oxo; and    -   R^(a) is hydrogen or C₁-C₈ alkyl.

In some embodiments, Y can be chloro; Z can be —NR¹(R²), in which R¹ andR², in some instances, may be C₁-C₈ alkyl (e.g., methyl); and/or R³ canbe OR^(a), in which R^(a) may be H in some instances. In some examples,R³ can be halo (e.g., chloro). In some examples, R⁴ can be hydrogen. Inother examples, R⁴ can be oxo.

In some embodiments, the compound may have one of the followingstructures:

Further, the present disclosure provides a compound having the followingstructure (I):

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof,

wherein:

-   -   A represents a 6-membered aromatic ring or a 6-membered        carbocyclic ring;    -   R^(1a) and R^(1b) are each independently H, C1-C₆ alkyl, or —OH;    -   R² and R³ are each independently H, C1-C₆ alkyl, or halo;    -   R⁴ is H, C1-C₆ alkyl, or —OH;    -   R^(5a) and R^(5b) are each independently H, C1-C₆ alkyl, or        halo;    -   R^(6a), R^(6b), R^(6c) and R^(6d) are each independently absent        or —O⁻;    -   R⁷ is H, C1-C₆ alkyl, —OH or absent;    -   R⁸ is absent or has the following structure:

-   -   R⁹ is absent or alkenyl, provided that at least one of R⁸ or R⁹        is present;    -   R¹⁰ is H or C1-C₆ alkyl; and    -   represents a double or single bond; and    -   all valencies are satisfied;    -   provided that if R^(1a) and R^(1b) are both methyl, then:    -   a. at least one of R^(6a), R^(6b), R^(6c), R^(6d) and R⁹ is —O⁻;    -   b. R⁷ is C1-C₆ alkyl, —OH or absent; and/or    -   c. R¹⁰ is H.

In some embodiments, the compound may be one of the following:

or pharmaceutically acceptable salts thereof.

Further, the present disclosure provides a pharmaceutical compositioncomprising any of the compounds disclosed herein, or a stereoisomer,pharmaceutically acceptable salt, tautomer or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.

Also within the scope of the present disclosure is a method of treatingcancer in a subject, the method comprising administering an effectiveamount of any of the compounds disclosed herein, or a stereoisomer,pharmaceutically acceptable salt, tautomer or prodrug thereof, or apharmaceutical composition as also disclosed herein.

Further, provided herein is a method for determining a metabolic profileof a subject, the method comprising: contacting a population of cells ofthe subject with a therapeutic agent; and determining a concentration ofa first metabolite that is any of the compounds disclosed herein. Insome embodiments, the therapeutic agent can be a compound that has thefollowing structure (VI):

or a pharmaceutically acceptable salt or crystalline form thereof. Insome embodiments, the cancer is melanoma (e.g., metastatic melanoma),breast cancer (e.g., inflammatory breast cancer and/or triple negativebreast cancer), or brain tumor (e.g., gliblastoma multiforme (GBM)). Insome embodiments, the subject shows disease progression, recurrence, orrelapse after treatment with an immunotherapy.

The method disclosed herein may further comprise administering animmunotherapeutic agent to the subject. In some embodiments, theimmunotherapy is a checkpoint inhibitor or a CTLA-4 inhibitor. Forexample, the immunotherapeutic agent is pembrolizumab. In someembodiments, the method can be performed under one or more of theconditions disclosed herein.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are enlarged andpositioned to improve figure legibility. Further, the particular shapesof the elements as drawn are not intended to convey any informationregarding the actual shape of the particular elements, and have beensolely selected for ease of recognition in the figures.

FIG. 1 illustrates PK results comparing salt forms of the compound ofstructure (I).

FIG. 2 shows the mean plasma concentration of a compound of structure(I) when administered as a free base and as a tartrate salt.

FIG. 3 provides cell viability data for combinations of a CDK inhibitorand an AXL kinase inhibitor in DOHH2 cells.

FIG. 4 is additional cell viability for combinations of a CDK inhibitorand an AXL kinase inhibitor in HCT-116 cells.

FIG. 5 shows an increased number of active DCs within tumors followingadministration of an AXL kinase inhibitor.

FIG. 6A shows tumor volume and FIG. 6B shows and body weight followingtreatment with an AXL kinase inhibitor and a PD-L1 antibody in the 4T-1breast cancer syngraft in mice.

FIG. 7 shows tumor volume following treatment with an AXL kinaseinhibitor and a PD1 antibody, with or without radiation therapy, in amouse breast cancer model.

FIG. 8 shows tumor volume following treatment with an AXL kinaseinhibitor and a PD1 antibody, with or without radiation therapy, in amouse melanoma model.

FIG. 9 shows tumor volume following treatment with an AXL kinaseinhibitor and a PD1 antibody in a mouse colorectal cancer model.

FIG. 10 shows tumor volume following treatment with an AXL kinaseinhibitor and a PD1 antibody in a mouse lung cancer model.

FIG. 11 shows activity of a compound of structure (I) in a syngeneicmouse model for breast cancer. FIG. 11A shows tumor volumes and FIG. 11Bshows body weights for animals on study.

FIG. 12 shows tumor volume following treatment with an AXL kinaseinhibitor and an EGFR inhibitor in an EGFR mutated lung cancer model.

FIG. 13 shows tumor volume following treatment with an AXL kinaseinhibitor and two different EGFR inhibitors in an EGFR mutated lungcancer model.

FIG. 14 shows protein expression levels of pAXL and total AXL.

FIG. 15A and FIG. 15B show changes in EMT marker expression aftertreatment with a compound of structure (I).

FIG. 16A and FIG. 16B show effects of treatment with a compound ofstructure (I) on the migration of Panc-1 or Aspc-1 cells.

FIG. 17A and FIG. 17B show treatment effects of a compound of structure(I), in combination with erlotinib, in an in vivo xenograft model forlung cancer.

FIG. 18A and FIG. 18B show treatment effects of a compound of structure(I), in combination with erlotinib, in an in vivo xenograft model forlung cancer.

FIG. 18C and FIG. 18D show the compound of structure (I) and EGFRiactivity in the H1650 NSCLC cell line. H1650 cells were incubated in thepresence of the indicated drugs for 72 hours, following which cellviability was assessed using the CellTiter-Glo reagent according tomanufacturer protocol. H1650 cells treated with single-agents (IC₅₀):the compound of structure (I) (35.9 nM), erlotinib (9.9 μM), orosimertinib (2.2 μM) (FIG. 18C). H1650 cells were treated withcombinations of the compound of structure (I) and erlotinib (FIG. 18D).

FIG. 18E and FIG. 18F shows combination activity of the compound ofstructure (I) and erlotinib in the H1650 xenograft model. H1650xenograft tumor bearing mice were treated daily by oral gavage witheither the compound of structure (I) (40 mg/kg), erlotinib (20 mpk), orthe combination. Tumor volumes and bodyweights were assessed twiceweekly.

FIG. 18G and FIG. 18H show combination activity of the compound ofstructure (I) and osimertinib in the H1650 xenograft model. H1650xenograft tumor bearing mice were treated daily by oral gavage witheither the compound of structure (I) (40 mg/kg), osimertinib (20 mpk),or the combination. Tumor volumes and bodyweights were assessed twiceweekly.

FIG. 19A and FIG. 19B show efferocytosis assays interrogating theeffects of a compound of structure (I) treatment in phorbol ester (PMA)treated THP-1 cells (macrophage induction).

FIG. 20 shows CYP26A1 mRNA levels of RA in cells treated with a compoundof structure (I).

FIG. 21A and FIG. 21B show CYP26A1 mRNA expression in MV4-11 cells.

FIG. 22A and FIG. 22B show AXL co-immunoprecipitates with RA importassociated gene, Stra6.

FIG. 23 shows protein levels of pAXL and total AXL.

FIG. 24 shows a compound of structure (I) inhibits basal expression ofCYP26A1.

FIG. 25 shows changes in CYP26A1 expression over 24 hours.

FIG. 26 shows CYP26A1 expression in R428 treated cells.

FIG. 27 shows changes in CYP26A1 expression and intracellular RA levels.

FIG. 28 shows that a compound of structure (I) affects the expression ofmultiple genes involved in RA synthesis and metabolism.

FIG. 29 shows a selected list of genes that respond to RA and a compoundof structure (I).

FIG. 30A and FIG. 30B show CYP26A1 mRNA expression in (FIG. 30A) MV4-11and (FIG. 30B) A549 cells.

FIG. 31 shows CYP26A1 protein expression in A549 cells treated with RA.

FIG. 32 shows changes in CYP26A1 expression over 72 hours with low-dosetreatment with a compound of structure (I).

FIG. 33 shows changes in CYP26A1 expression over 24 hours for MV4-11cells treated with R428.

FIGS. 34A-34D show (FIGS. 34A/34B) MV4-11 and (FIGS. 34C/34D) A549xenograft studies.

FIG. 35 is a photo showing changes in CYP26A1 expression in vivo beforeand after treatment.

FIG. 36 is a chart showing fold changes of CYP26 in tumors over time inmice treated with compound of structure (I) as relative to untreatedmice.

FIG. 37 is a chart showing fold changes of CYP26 in livers over time inmice treated with compound of structure (I) as relative to untreatedmice.

FIGS. 38-40 show serum levels of GAS6 (FIG. 38); and GAS6 and AXL (FIGS.39 and 40) at the following times: Cycle 1/Day 1 pre-dose (“C1D1 PRE”),and 2 and 24 hours post-dose (“C1D1 2” and “C1D1 24”, respectively) andCycle 1/Day 8 pre-dose (“C1D8 PRE”).

FIG. 41 shows plasma levels of AXL and GAS6 at day twenty (collectedfour hours post-dosing) of treatment with the compound of structure (I),in a patient-derived xenograft model of colorectal cancer.

FIG. 42 shows mRNA expression levels of epithelial-to-mesenchymaltransition markers at day 20 of treatment with the compound of structure(I), in a patient-derived xenograft model of colorectal cancer.

FIG. 43 shows mRNA expression levels of epithelial-to-mesenchymaltransition markers at day 27 (one week after the last dosing) oftreatment with the compound of structure (I), in a patient-derivedxenograft model of colorectal cancer.

FIG. 44 shows mRNA expression levels of markers of activated dendriticcells (CD86 and CD11c), at day 20 (top panel) and day 27 (one week afterthe last dosing, bottom panel) of treatment with the compound ofstructure (I), in a patient-derived xenograft model of colorectalcancer.

FIG. 45A shows the baseline AXL levels in serum from patients withprogressive disease (PD) versus stable disease (SD).

FIG. 45B shows the baseline AXL levels in serum from patients withprogressive disease (PD) versus stable disease (SD).

FIG. 46A shows the baseline GAS6 levels in serum from patients with PDversus SD.

FIG. 46B shows the baseline GAS6 levels in serum from patients with PDversus SD.

FIGS. 47A-47C show the sensitivity of regulatory T-cells to the compoundof structure (I).

FIG. 48 shows total tumor infiltrating lymphocytes (TILs) found insamples treated with vehicle or 25 milligrams (mg)/kilogram (kg) of acompound of structure (I).

FIG. 49 shows dendritic cells found in samples from a 4T1 model treatedwith vehicle or 25 mg/kg of a compound of structure (I).

FIG. 50 shows macrophages found in samples from a 4T1 model treated withvehicle or 25 mg/kg of a compound of structure (I).

FIG. 51 shows neutrophils found in samples from a 4T1 model treated withvehicle or 25 mg/kg of a compound of structure (I).

FIG. 52 shows natural killer (NK) cells found in samples from a 4T1model treated with vehicle or 25 mg/kg of a compound of structure (I).

FIG. 53 shows regulatory T cells (Tregs) found in samples from a 4T1model treated with vehicle or 25 mg/kg of a compound of structure (I).

FIG. 54 shows exhausted CD8 T cells found in samples from a 4T1 modeltreated with vehicle or 25 mg/kg of a compound of structure (I).

FIG. 55 shows the combination effect of the compound of structure (I)with anti-PD-1 in a 4T1 model.

FIG. 56A and FIG. 56B show the anti-tumor effect of the compound ofstructure (I) in combination with PD-1 in CD8+ T depletion mice.

FIG. 57 shows effects of the compound of structure (I) on immune cellsin the spleen (depicted as total cells in spleen at Day16).

FIGS. 58A and 58B show effects of the compound of structure (I) onimmune cells in tumors (depicted as cells/mg tumors at Day11).

FIG. 59 shows effects of the compound of structure (I) on geneexpression of cytokines and chemokines in tumors.

FIG. 60 shows a time course of regulation of cytokines and chemokines intumors following treatment with the compound of structure (I).

FIG. 61A illustrates an x-ray diffractogram obtained from XRPD analysisfor crystalline Form A.

FIG. 61B illustrates an ¹HNMR spectrum of crystalline Form A.

FIG. 61C illustrates an ¹HNMR spectrum of crystalline Form A′.

FIG. 62A illustrates an x-ray diffractogram obtained from XRPD analysisfor crystalline Form B.

FIG. 62B illustrates an ¹HNMR spectrum of crystalline Form B.

FIG. 63A illustrates an x-ray diffractogram obtained from XRPD analysisfor polymorph Form D.

FIG. 63B illustrates an ¹HNMR spectrum of crystalline Form D.

FIG. 64 shows a TGA and DSC plot obtained for crystalline Form A.

FIG. 65 shows a comparison between Form B (upper) and Form A (lower).

FIG. 66 shows the effects on cell viability of ES-2 cells treated with acompound of structure (I).

FIG. 67 shows the results of various concentrations of the compound ofstructure (I) on several chemotherapy-resistant ovarian cancer celllines (top right and top left panels), and the results of comparativetesting of the compound of structure (I), Cabozantinib, and Foretinib oncell viability of ovarian tumor cells in the platinum-resistantKuramochi cell line (bottom panel).

FIG. 68 compares the effects of the compound of structure (I) andCabozantinib on phospho-AXL (Y702) levels in the platinum-resistantKuramochi cell line.

FIG. 69 shows the results of epithelial to mesenchymal (EMT) markersthat were quantified by mRNA expression analysis two hours aftertreatment with the compound of structure (I) at various concentrations.

FIG. 70 shows the results of epithelial to mesenchymal (EMT) markersthat were quantified by mRNA expression analysis twenty-four hours aftertreatment with the compound of structure (I) at various concentrations.

FIG. 71 shows the protein expression levels measured for the EMT markersSnail and Slug following treatment with the compound of structure (I) atvarious concentrations.

FIG. 72 shows the effects of a compound of structure (I) and BMS-777607on the migration of ovarian tumor cells in a scratch assay.

FIG. 73 shows the circumference of the mice following treatment and arepresentative photo of a mouse in each treatment group, as described inExample 33.

FIG. 74 shows a graph of the body weight of mice following treatment, asdescribed in Example 33.

FIG. 75 shows the results of a panel of chemokines and cytokinesmeasured following treatment of ES-2 xenograft mice with a compound ofstructure (I).

FIG. 76 shows the mRNA expression levels of EMT markers assayed fromascites fluid following treatment with the compound of structure (I).

FIG. 77 shows soluble AXL, GAS6, and PD-L1 levels in blood followingtreatment with either 25 mg/kg or 50 mg/kg of the compound of structure(I), as described in Example 33.

FIG. 78 shows the relative levels of mouse versus human AXL, GAS6, andPD-L1 in blood, following treatment as described in Example 33.

FIG. 79 shows a representative photo of a mouse treated with a compoundof structure (I) versus a vehicle control (top left), body weight of thetreated mice, abdominal circumference of the treated mice, volume ofrecovered ascites, and percent survival, for mice treated as describedin Example 37.

FIG. 80 shows the results of western blots for tubulin, AXL total andphosphorylated at Y702 site and relative AXL phosphorylation of ES-2cell-injected mice compared to the control group, as described inExample 37.

FIG. 81 shows the abdominal circumference and body weight oftumor-bearing versus non-tumor bearing mice, for mice treated asdescribed in Example 39.

FIG. 82 shows the ascites volume and representative photographs of micetreated in the study described in Example 39, with non-tumor bearingmice shown on the left and tumor-bearing mice shown on the right.

FIG. 83 shows the results of treating the cells with varying doses ofthe compound of structure (I) as described in Example 40.

FIG. 84 shows the circumference of A2780cis xenograft mice 5-25 daysafter implant, as described in Example 40.

FIG. 85 shows the bodyweight of A2780cis xenograft mice 5-25 days afterimplant, as described in Example 40.

FIG. 86 shows representative photographs of mice in the study at day 25after treatment, as described in Example 40.

FIG. 87 shows gene expression levels in tumors of A2780cis xenograftmice, as described in Example 40.

FIG. 88 shows protein expression levels were measured by western blotanalysis for Snail and β-actin as a loading control following treatmentwith the compound of structure (I), as described in Example 40.

FIG. 89 shows human Axl expression in serum, as described in Example 40.

FIGS. 90-92 shows representative data for Compound IV plasmaconcentrations on day 1 and day 21 from subjects dosed for 21consecutive days with Compound VI.

FIG. 93 provides Compound VI and active metabolite data collected andshown as area under the curve (AUC) values for day 1 and day 21 dosingwith Compound VI for 21 consecutive days.

FIG. 94 shows XRPD peaks characteristic of Form B.

FIG. 95 shows TGA/DSC curves of Form B.

FIG. 96 shows XRPD peaks characteristic of Form D.

FIG. 97 shows TGA/DSC curves of Form D.

FIG. 98A shows a comparison between XRPD diffractograms of crystallineForms A, B, and C.

FIG. 98B shows a comparison between XRPD diffractograms of crystallineForms D, E, F, G, H, and I.

FIGS. 99A-99I shows thermal behavior (DSC/TGA charts) for crystallineForms A through I.

FIG. 100 shows a general scheme outling how the compound of structure(I) undergoes metabolic conversion into a number of species.

FIGS. 101A-101E show ¹H (proton) NMR spectra for metabolites M2, M3, M4,M6, and M7.

FIGS. 102A-102B show relative amounts of active metabolites after 21days in two different cohorts.

FIGS. 103A-103D show ¹H (proton) NMR spectra for intermediates in thesynthesis of the compound of structure (I).

FIGS. 104A-104B illustrate that the compound of structure (I) inhibitscell growth of colorectal carcinoma (CRC) cells lines independent ofKRAS mutation status. FIG. 104A shows KRAS mutation status of selectedCRC cell lines. FIG. 104B shows CRC cell viability determinationfollowing 72 hrs treatment with the compound of structure (I) andassessment via CellTiter-Glo. The compound of structure (I) demonstratedpotency independent of the KRAS mutation status with IC₅₀ between 4.5 nMand 123 nM.

FIGS. 105A-105B shows that the compound of structure (I) suppressesmesenchymal markers without modulating epithelial markers. HCT-116 cellswere treated with indicated concentrations of AXL inhibitors: R428,RXDX-106, and the compound of structure (I) for 24 hrs. FIG. 105A showsmRNA expression levels were quantified via RT-qPCR. FIG. 105B showsprotein expression levels were analyzed via western blot. Snailexpression was suppressed by 7.6 fold (m-RNA) and 4.9 fold (protein)with 500 nM compound of structure (I).

FIGS. 106A-106C shows the compound of structure (I) inhibits tumorgrowth in the KRAS mutant HCT-116 xenograft model. Athymic nude micewere injected in the hind-flank with 10 million cells and stratifiedinto cohorts of 10 mice. Compounds were formulated in 5% (w/v) TPGS and1% (v/v) PS80 in H20 and administered my oral gavage. Tumor volumes(FIG. 106A) and body weights were measured twice a week (FIG. 106B).Intra-tumoral GAS6 expression was quantified via RT-qPCR. The 40 mg/kgcompound of structure (I) cohort achieved 69% TGI without adverse events(FIG. 106C).

FIGS. 107A-107B show the compound of structure (I) inhibits tumor growthin the KRAS mutant PDX CRC model. Balb/c nude mice were implanted with2-3 mm fragments of primary human CRC tumors and then stratified intocohorts of 10 mice. The compound of structure (I) was formulated in 5%(w/v) TPGS and 1% (v/v) PS80 in H20 and administered by oral gavage.Tumor volume (FIG. 107A) and bodyweights (FIG. 107B) were measured twicea week. The 40 mg/kg compound of structure (I) cohortachieved 44% TGIwithout adverse events.

FIGS. 108A-108E show the compound of structure (I) suppresses sAXL/sGAS6concentrations while down-regulating Wnt/β-catenin regulatedgenesAxin2/CCND1 in the KRAS mutant PDX CRC model. sAXL (FIG. 108A) andsGAS6 (FIG. 108B) were quantified in the serum via ELISA. Intra-tumoralGAS6 (FIG. 108C), Axin2 (FIG. 108D), and CCND1 (FIG. 108E) werequantified via RT-qPCR. Analyses were performed on mice treated for 27days (except Axin2; 21 days). Suppression of sAXL and sGAS6 indicate areversal of EMT. The compound of structure (I)-mediated downregulationof Wnt/β-catenin associated genes further supports a previously reportedrole for AXL in β-catenin stabilization.

FIG. 109 shows a positive correlation between sAXL/sGAS6 and tumorvolume in the KRAS mutant PDX CRC model identifies them as potentialbiomarkers for disease progression. Soluble concentrations werequantified in each mouse's serum via ELISA followed linear regressionanalysis. Statistical significance for each correlation: sAXL and tumorvolume (P<0.005); and sGAS6 and tumor volume (P<0.0005).

FIGS. 110A-110B shows EMT marker expression in the compound of structure(I)-treated NSCLC cells. H1650 (FIG. 110A) and A549 cells (FIG. 110B)were treated for two hours with the compound of structure (I) atconcentrations up to 2 μM, following which snail and slug mRNAexpression was assessed using standard qPCR technique.

FIGS. 111A-111B show EMT marker protein expression in AXLinhibitor-treated H1650 and A549 cells. H1650 cells were treated with0.1, 0.5, or 1.0 μM of the compound of structure (I) or R428, for 24hours, following which cells were harvested and E-cadherin and snailprotein expression was assessed using standard western immunblottingtechnique (FIG. 111A). A549 cells were treated with 0.1, 0.5, or 1.0 μMcompound of structure (I) or R428, for 24 hours, following which cellswere harvested and E-cadherin and Snail protein expression was assessedusing standard western immunblotting technique (FIG. 111B).

FIG. 112 shows slug mRNA expression in the compound of structure (I)treated H1650 xenograft mice. H1650 xenograft tumor bearing mice weretreated with the compound of structure (I) (40 mg/kg) by oral gavage,following which tumors were harvested at varying timepoints followingdosing. Slug and E-cadherin mRNA expression was assessed by standardqPCR technique.

FIG. 113 shows snail protein expression in the compound of structure(I)-treated H1650 xenograft mice. H1650 xenograft tumor bearing micewere treated with the compound of structure (I) (40 mg/kg) by oralgavage, following which tumors were harvested at varying timepointsfollowing dosing. Slug and E-cadherin protein expression was assessed bystandard immunoblotting technique.

FIG. 114 shows pharmacokinetic profile of the compound of structure (I)in 4T1 model. 4T1 bearing mice were treated with 60 mpk of the compoundof structure (I) tartrate p.o. Tumor and blood were collected at theindicated time points.

FIG. 115 shows effect of the compound of structure (I) on cytokines inserum. Balb/c mice were transplanted with 4T1 cells orthotopically. 7days after transplantation, the compound of structure (I) tartrate wasadministrated (60 mg/kg, p.o., Q.D.). Whole blood was collected 2, 6,and 24 hour after the last dosage on Day12. Cytokines in serum weremeasured with Milliplex assay. Normal indicates healthy mouse withouttumor, n=6 (vehicle: n=5, normal: n=3). Error Bar indicates SD. N.Dindicates “no data”.

FIG. 116 shows moisture sorption isotherms for Forms A′, A, B, C, and D.

FIG. 117 shows the comparison of XRPD patterns of Form A and Form Dbetween before and after moisture sorption isotherm.

FIG. 118 shows Raman spectra and its PCA data for Form A and Form B.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense (i.e., as “including, but not limited to”).

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an”, and “the” include plural referencesunless the context clearly dictates otherwise.

“Oxo” refers to the ═O substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C1-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C1-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Alkyl includes alkenyls (one or more carbon-carbon double bonds)and alkynyls (one or more carbon-carbon triple bonds such as ethynyl andthe like). Unless stated otherwise specifically in the specification, analkyl group is optionally substituted.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

The term “substituted” as used herein means at least one hydrogen atomis replaced by a bond to a non-hydrogen atoms such as, but not limitedto: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groupssuch as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atomin groups such as thiol groups, thioalkyl groups, sulfone groups,sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such asamines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines,diarylamines, N-oxides, imides, and enamines; a silicon atom in groupssuch as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilylgroups, and triarylsilyl groups; and other heteroatoms in various othergroups. “Substituted” also means one or more hydrogen atoms are replacedby a higher-order bond (e.g., a double- or triple-bond) to a heteroatomsuch as oxygen in oxo, carbonyl, carboxyl, and ester groups; andnitrogen in groups such as imines, oximes, hydrazones, and nitriles. Forexample, in some embodiments “substituted” means one or more hydrogenatoms are replaced with —NR_(g)R_(h), —NR_(g)C(═O)R_(h),—NR_(g)C(═O)NR_(g)R_(h),

—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted also means one or more hydrogen atoms arereplaced with —C(═O)R_(g), —C(═O)OR_(g), —C(═O)NR_(g)R_(h),—CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). In the foregoing, R_(g) and R_(h) arethe same or different and independently hydrogen, alkyl, alkoxy,alkylaminyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl. “Substituted” further means one ormore hydrogen atoms are replaced by a bond to an aminyl, cyano,hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylaminyl,thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl,heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl group. In addition, each of theforegoing substituents may also be optionally substituted with one ormore of the above substituents.

The term “effective amount” or “therapeutically effective amount” refersto that amount of compound (e.g., a compound of structure (I) or apharmaceutically acceptable salt, such as a tartrate salt, of thecompound of structure (I)) described herein that is sufficient to effectthe intended application including but not limited to disease treatment,as defined below. The therapeutically effective amount may varydepending upon the intended treatment application (in vivo), or thesubject and disease condition being treated, e.g., the weight and age ofthe subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, e.g., reduction ofplatelet adhesion and/or cell migration. The specific dose will varydepending on the particular compounds chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to which it isadministered, and the physical delivery system in which it is carried.

A “cancer,” including a “tumor,” refers to an uncontrolled growth ofcells and/or abnormal increased cell survival and/or inhibition ofapoptosis which interferes with the normal functioning of the bodilyorgans and systems. “Cancer” (e.g., a tumor) includes solid andnon-solid cancers. A subject that has a cancer or a tumor has anobjectively measurable number of cancer cells present in the subject'sbody. “Cancers” include benign and malignant cancers (e.g., benign andmalignant tumors, respectively), as well as dormant tumors ormicrometastases.

A cancer that is “resistant” to a particular therapy refers to a cancerthat demonstrates persistent disease or complete remission for less than6 months after administration of the therapy. In some embodiments, asubject that has a cancer that is resistant to a particular therapyshows no statistically significant objective response to the therapy. Asubject is considered to be in “complete remission” if the subject has anormal CA-125 level (e.g., less than 46 U/mL) and a normal CT scan. Insome embodiments, a cancer is resistant to a particular therapy if thecancer progresses while receiving the therapy or within six months ofthe last administration of the therapy.

A recurrent cancer refers to a cancer that appears in a site where itwas eradicated or disappeared. A treatment resistant cancer (e.g.,platinum-resistant) is “recurrent” if the cancer has progressed (e.g.,confirmed by imaging) if the subject had recurrence within six months ofthe last receipt of the treatment. For example, platinum-resistantcancer is recurrent if the subject had recurrence within 6 months of thelast receipt of platinum-based chemotherapy.

As used herein, the term “refractory” with respect to a subject havingAML has its ordinary meaning in the art and may refer to a subject thathas residual leukemic cells in their marrow after treatment, e.g.,within one week, within two weeks, within four weeks, or within twomonths after treatment.

A cancer is “persistent” if the cancer exists or remains in the samestate for an indefinitely long time.

As used herein, the term “relapse” has its ordinary meaning in the artand may refer to the return of AML or the signs and symptoms of an AMLafter a period of complete remission (e.g., initial complete remission)due to treatment. In some embodiments, relapse may refer to therecurrence of disease after complete remission, which a may bedetermined by a physician upon clinical assessment.

A cancer is “partially sensitive” to a therapy (e.g., partially platinumsensitive) if the cancer progresses between 6 and 12 months after thelast administration of the therapy.

A cancer is “sensitive” to a therapy (e.g., platinum sensitive) if thecancer progresses more than 12 months after the last administration ofthe therapy. “Metastasis” refers to the spread of cancer from itsprimary site to other places in the body. “Metastases” are cancers whichmigrate from their original location and seed vital organs, which caneventually lead to the death of the subject through the functionaldeterioration of the affected organs. Metastasis is a sequentialprocess, where cancer cells can break away from a primary tumor,penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. At the new site, the cells establish a bloodsupply and can grow to form a life-threatening mass. Metastasis can belocal or distant. Both stimulatory and inhibitory molecular pathwayswithin the tumor cell regulate this behavior, and interactions betweenthe tumor cell and host cells in the new site are also significant.

A subject having at least “stable disease” refers to a subject havingstable disease, partial response or complete response to a therapyaccording to iRECIST.

As used herein, “treatment” or “treating” refers to an approach forobtaining beneficial or desired results with respect to a disease,disorder or medical condition including but not limited to a therapeuticbenefit and/or a prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated.Also, a therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thesubject, notwithstanding that the subject may still be afflicted withthe underlying disorder. In certain embodiments, for prophylacticbenefit, the compositions are administered to a subject at risk ofdeveloping a particular disease, or to a subject reporting one or moreof the physiological symptoms of a disease, even though a diagnosis ofthis disease may not have been made.

As used herein, the term “treatment cycle” has its ordinary meaning inthe art and may refer to one or more course of treatments that arerepeated on a regular schedule, including periods of rest.

A “therapeutic effect,” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

The term “co-administration,” “administered in combination with,” andtheir grammatical equivalents, as used herein, encompass administrationof two or more agents to an animal, including humans, so that bothagents and/or their metabolites are present in the subject at the sametime. Co-administration includes simultaneous administration in separatecompositions, administration at different times in separatecompositions, or administration in a composition in which both agentsare present.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid (e.g., L-(+)-tartaric acid), thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, A-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

In some embodiments, pharmaceutically acceptable salts includequaternary ammonium salts such as quaternary amine alkyl halide salts(e.g., methyl bromide).

An “anti-cancer agent”, “anti-tumor agent” or “chemotherapeutic agent”refers to any agent useful in the treatment of a neoplastic condition.One class of anti-cancer agents comprises chemotherapeutic agents.“Chemotherapy” means the administration of one or more chemotherapeuticdrugs and/or other agents to a cancer patient by various methods,including intravenous, oral, intramuscular, intraperitoneal,intravesical, subcutaneous, transdermal, buccal, or inhalation or in theform of a suppository.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both human therapeuticsand veterinary applications. In some embodiments, the subject is amammal, and in some embodiments, the subject is human.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Radiation therapy” means exposing a subject, using routine methods andcompositions known to the practitioner, to radiation emitters such asalpha-particle emitting radionuclides (e.g., actinium and thoriumradionuclides), low linear energy transfer (LET) radiation emitters(i.e., beta emitters), conversion electron emitters (e.g., strontium-89and samarium-153-EDTMP, or high-energy radiation, including withoutlimitation x-rays, gamma rays, and neutrons.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically active saltdescribed herein (e.g., the tartrate salt of structure (I)). Thus, theterm “prodrug” refers to a precursor of a biologically active compoundthat is pharmaceutically acceptable. In some aspects, a prodrug isinactive when administered to a subject, but is converted in vivo to anactive compound, for example, by hydrolysis. The prodrug compound oftenoffers advantages of solubility, tissue compatibility or delayed releasein a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs(1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion of prodrugsis provided in Higuchi, T., et al., “Pro-drugs as Novel DeliverySystems,” A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporated infull by reference herein. The term “prodrug” is also meant to includeany covalently bonded carriers, which release the active compound invivo when such prodrug is administered to a mammalian subject. Prodrugsof an active compound, as described herein, are typically prepared bymodifying functional groups present in the active compound in such a waythat the modifications are cleaved, either in routine manipulation or invivo, to the parent active compound. Prodrugs include compounds whereina hydroxy, amino or mercapto group is bonded to any group that, when theprodrug of the active compound is administered to a mammalian subject,cleaves to form a free hydroxy, free amino or free mercapto group,respectively. Examples of prodrugs include, but are not limited to,acetate, formate and benzoate derivatives of a hydroxy functional group,or acetamide, formamide and benzamide derivatives of an amine functionalgroup in the active compound and the like.

The term “m vivo” refers to an event that takes place in a subject'sbody.

“About” and “approximately,” when used in connection with a numericvalue or range of values which is provided to describe a particularsolid form, e.g., a specific temperature or temperature range, such as,for example, that describing a melting, dehydration, desolvation orglass transition; a mass change, such as, for example, a mass change asa function of temperature or humidity; a solvent or water content, interms of, for example, mass or a percentage; or a peak position, suchas, for example, in analysis by IR or Raman spectroscopy or XRPD;indicate that the value or range of values may deviate to an extentdeemed reasonable to one of ordinary skill in the art while stilldescribing the particular solid state form. Specifically, the terms“about” and “approximately,” when used in this context, indicate thatthe numeric value or range of values may vary by 20%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%,0.1% or 0.01% of the recited value or range of values while stilldescribing the particular composition or solid state form.

“Substantially identical” as used herein refers to measured physicalcharacteristics that are comparable in value or data traces that arecomparable in peak position and amplitude or intensity within the scopeof variations that are typically associated with sample positioning orhandling or the identity of the instrument employed to acquire thetraces or physical characteristics or due to other variations orfluctuations normally encountered within or between laboratoryenvironments or analytical instrumentation.

“Substantially pure” as used herein refers to a solid state form of acompound described herein that contains less than about 3% or less thanabout 2% by weight total impurities, or more preferably less than about1% by weight water, and/or less than about 0.5% by weight impuritiessuch as decomposition or synthesis by-products or residual organicsolvent.

“Essentially pure” as used herein refers to a form of a compounddescribed herein wherein the sum of impurities or related substance inthe form is less than 1%, preferably less than 0.75%, more preferablyless than 0.5% and that the residual solvents and water are less than1%, preferably less than 0.75%, more preferably less than 0.5% and stillmore preferably less than 0.25% by weight.

The term “crystalline forms” and related terms herein refers to thevarious crystalline states of a given substance, including, but notlimited to, polymorphs, solvates, hydrates, mixed solvates, co-crystalsand other molecular complexes. A crystalline form may also be, but isnot necessarily, a mixture of various crystalline states of a givensubstance such as a combination of pseudopolymorph or polymorph forms, acombination of one or more polymorph forms with one or morepseudopolymorph or a combination of such forms with amorphous ornon-solid state forms of the substance. Typical combinations are of twoor more polymorph or pseudo polymorph forms, such a mixture of apolymorph form with a pseudopolymorph form or a mixture of a polymorphor pseudopolymorph form with amorphous material. Typically crystallineforms are typically distinguishable from each other by their XRPDpatterns. Solid state forms having different crystal morphologies butessentially identical XRPD patterns are considered to be differentcrystalline forms, since different morphologies can exhibit differentproperties related to physical shape. Properties related to physicalshape include dissolution rate, stability, hygroscopicity, mechanicalproperties such hardness, tensile strength, compatibility (tableting)and those related to handling, e.g., flow, filtering, blending and otherphysical or pharmaceutical properties as described herein for differentpolymorphs.

Embodiments of the invention disclosed herein are also meant toencompass pharmaceutically acceptable salts of a compound of structure(I) being isotopically-labelled by having one or more atoms replaced byan atom having a different atomic mass or mass number (i.e., an“isotopic form” of the pharmaceutically acceptable salts of a compoundof structure (I)). Examples of isotopes that can be incorporated intothe disclosed compounds include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H,¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶C, ¹²³I,and ¹²⁵I, respectively. These radiolabeled compounds could be useful tohelp determine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to pharmacologically important site of action. Certainisotopically-labeled pharmaceutically acceptable salts of compounds ofstructure (I), for example, those incorporating a radioactive isotope,are useful in drug and/or substrate tissue distribution studies. Theradioactive isotopes tritium (i.e. ³H), and carbon-14 (i.e., ¹⁴C) areparticularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium (i.e., ²H) mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence are preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled salts ofcompounds of structure (I) can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Examples as set out below using an appropriateisotopically-labeled reagent in place of the non-labeled reagentpreviously employed.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds. “Stable compound”and “stable structure” are meant to indicate a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Zwitterionic form” refers to a form of a compound (e.g. structure(II′)), wherein at least one functional group has a positive charge, onefunctional group has a negative electrical charge, and the net charge ofthe entire molecule is zero. For example, a phosphate group (—PO₃H₂) mayexist in an anionic form (e.g., —PO₃H⁻), and a nitrogen atom within thesame molecule may exist in the protonated (cationic form).

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. Embodiments thus include tautomers ofthe disclosed compounds.

A “unit dose” is a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage, such as one-half or one-third of such adosage.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0.1software naming program (CambridgeSoft). For complex chemical namesemployed herein, a substituent group is typically named before the groupto which it attaches. For example, cyclopropylethyl comprises an ethylbackbone with a cyclopropyl substituent. Except as described below, allbonds are identified in the chemical structure diagrams herein, exceptfor all bonds on some carbon atoms, which are assumed to be bonded tosufficient hydrogen atoms to complete the valency.

B. Tartrate Salts of Compound of Structure (I)

In one aspect, the present disclosure provides a tartrate salt of acompound of structure (I):

In certain embodiments, the tartrate salt is a salt of L-(+)-tartaricacid. In certain particular embodiments, the tartrate salt of thecompound of structure (I) is a crystalline or partially crystallinesolid.

Other salt forms of the compound of structure (I) are provided. The saltmay be a pharmaceutically acceptable salt. The pharmaceuticallyacceptable salt of a compound of structure (I) can be represented by thefollowing structure:

wherein B− is the conjugate base of the acid used for salt formation. Incertain embodiments, the pharmaceutically acceptable salt is aphosphoric acid salt. In certain embodiments, the pharmaceuticallyacceptable salt is a malate salt. In certain embodiments, thepharmaceutically acceptable salt is a succinate salt. In certainembodiments, the pharmaceutically acceptable salt is a benzenesulfonatesalt.

Several salts of the compound of structure (I) were screened, asdescribed in Example 1. The tartrate salt exhibits favorablepharmacokinetic properties, such as bioavailability, as described inExamples 2 and 3.

1. Stoichiometric and Crystalline Forms

In some embodiments, the molar ratio of tartaric acid to the compound ofstructure (I) ranges from about 4:1 to about 1:4, from about 3.5:1 toabout 1:3.5, from about 3.2:1 to about 1:3.2, from about 3:1 to about1:3, from about 2.7:1 to about 1:2.7, from about 2.5:1 to about 1:2.5,from about 2.2:1 to about 1:2.2, from about 2:1 to about 1:2.2, fromabout 1.8:1 to about 1:2.2, from about 1.5:1 to about 1:2.2, from about1.2:1 to about 1:2.2, from about 1.1:1 to about 1:2.2, from about 0.8:1to about 1:2.2, from about 0.5:1 to about 1:2.2, from about 0.2:1 toabout 1:2.2, from about 0.1:1 to about 1:2.2, or from about 2:1 to about1:2.5.

In certain embodiments, the molar ratio of tartaric acid to the compoundof structure (I) is about 1:1; e.g., from about 0.8:1 to about 1.2:1. Incertain embodiments, the molar ratio is 0.8:1, 0.9:1, 1:1, 1.1:1, or1.2:1. In a particular embodiment, the molar ratio is 1:1. In anotherparticular embodiment, the molar ratio is 1.2:1. A specific embodimentprovides a tartrate salt having the following structure (IIa):

In certain embodiments, the tartrate salt of a compound of structure (I)has one of the following structures (IIb), (IIe), (IId), (IIe), (IIf) or(IIg):

a. Form B

In certain embodiments, a tartrate salt having a stoichiometry of about1:1 is of crystalline Form B, and is characterized by one or more ofX-ray powder diffraction (XRPD), Differential Scanning Calorimetry(DSC), and Thermogravimetric Analysis (TGA). In a particular embodiment,Form B has a molar ratio of tartaric acid to the compound of Structure(I) of 1:1. In another particular embodiment, Form B has a molar ratioof tartaric acid to the compound of Structure (I) of 1.2:1.

In certain embodiments, Form B is characterized by an XRPD patterncomprising two or more peaks, in units of 2-theta, selected from7.5±0.2, 10.3±0.2, 18.9±0.2, and 19.0±0.2 at a temperature of about 22°C. In certain embodiments, the XRPD pattern of Form B comprises 2, 3, or4 peaks selected from 7.5±0.2, 10.3±0.2, 18.9±0.2, and 19.0±0.2. Incertain embodiments, the XRPD pattern is substantially identical to thatof FIG. 62. In certain embodiments, the XRPD pattern comprises one ormore (e.g., 1, 2, 3, 4, or 5) additional peaks selected from the peakslisted in FIG. 94.

In certain embodiments, Form B is characterized by a DSC thermogramcomprising an endotherm peak in units ° C. at about 101.9. In certainembodiments, the DSC thermogram comprises an endotherm peak in units °C. at about 140.1. In certain embodiments, the DSC thermogram comprisesendotherm peaks in units ° C. at about 101.9 and 140.1. In a particularembodiment, the DSC thermogram is substantially identical to that ofFIG. 95.

In certain embodiments, Form B is characterized by a TGA thermogramshowing weight loss of about 2.3% at 160° C. In a particular embodiment,the TGA thermogram is substantially identical to the thermogram shown inFIG. 95.

Physical and chemical properties of Form B are described in Example 26,Table 17. Toxicokinetic and toxicology profiles of Form B are describedin Example 27.

b. Form D

In certain embodiments, a tartrate salt having a stoichiometry of about1:1 is of crystalline Form D, and is characterized by one or more ofXRPD, DSC, and TGA. In a particular embodiment, Form D has a molar ratioof tartaric acid to the compound of Structure (I) of 1:1.

In certain embodiments, Form D is characterized by an XRPD patterncomprising peaks, in units of 2-theta, at 12.8±0.2 and 18.9±0.2 at atemperature of about 22° C. In certain embodiments, the XRPD pattern issubstantially identical to that of FIG. 63. In certain embodiments, theXRPD pattern comprises one or more (e.g., 1, 2, 3, 4, or 5) additionalpeaks selected from the peaks listed in FIG. 96.

In certain embodiments, Form D is characterized an endotherm peak inunits ° C. at about 79.4. In certain embodiments, the DSC thermogramcomprises an endotherm peak in units ° C. at about 140.7. In certainembodiments, the DSC thermogram comprises endotherm peaks in units ° C.at about 79.4 and 140.7. the DSC thermogram is substantially identicalto that of FIG. 97.

In certain embodiments, Form D is characterized by a TGA thermogramshowing weight loss of about 2.0% at 160° C. In a particular embodiment,the TGA thermogram is substantially identical to that of FIG. 97.

In certain embodiments, a tartrate salt having a stoichiometry of about1:1 comprises Form D. In certain embodiments, a tartrate salt having astoichiometry of about 1:1 consists essentially of Form D. In certainembodiments, Form D is essentially pure.

Physical and chemical properties of Form B are described in Example 26,Table 17. Toxicokinetic and toxicology profiles of Form B are describedin Example 27.

c. Form A′

In certain embodiments, a tartrate salt having a stoichiometry of about1.5:1 is of crystalline Form A′, and is characterized by one or more ofXRPD, DSC, and TGA. In certain embodiments, the molar ratio of tartaricacid to the compound of structure (I) is about 1.5:1; e.g., from about1.4:1 to about 1.6:1. In certain embodiments, the molar ratio is 1.4:1,1.5:1, or 1.6:1. In a particular embodiment, the molar ratio is 1.5:1.In certain embodiments, crystalline Form A′ is characterized by a DSCthermogram comprising an endotherm peak at about 182.3° C. In certainembodiments, the endotherm peak has an onset temperature of about 170.5°C.

In certain embodiments, a tartrate salt having a stoichiometry of about1.5:1 comprises Form A′. In certain embodiments, a tartrate salt havinga stoichiometry of about 1.5:1 consists essentially of Form A′. Incertain embodiments, Form A′ is essentially pure.

d. Form A

In certain embodiments, a tartrate salt having a stoichiometry of about2:1 is of crystalline Form A, and is characterized by one or more ofXRPD, DSC, and TGA. In certain embodiments, the molar ratio of tartaricacid to the compound of structure (I) ranges from about 2.2:1 to about1.9:1. In a particular embodiment, the molar ratio is 2:1.

In certain embodiments, Form A is characterized by an XRPD patterncomprising three or more peaks, in units of 2-theta, selected from7.0±0.2, 11.2±0.2, 15.4±0.2, 16.3±0.2, 17.1±0.2, 19.9±0.2, 21.6±0.2, and25.5±0.2 at a temperature of about 22° C. In certain embodiments, theXRPD pattern of Form A comprises 3, 4, 5, 6, 7, or 8 peaks selected from7.0±0.2, 11.2±0.2, 15.4±0.2, 16.3±0.2, 17.1±0.2, 19.9±0.2, 21.6±0.2, and25.5±0.2. In certain embodiments, the XRPD pattern is substantiallyidentical to that of FIG. 61. In certain embodiments, the XRPD patterncomprises one or more (e.g., 1, 2, 3, 4, or 5) additional peaks selectedfrom the peaks listed in Table 1.

TABLE 1 Tabulated X-ray Powder Diffractogram data from Form A Net GrossRelative Angle D Value Intensity Intensity Intensity 7.016 12.58927 8721003 39.7% 7.137 12.37531 283 413 12.9% 7.671 11.51594 89.1 210 4.1%8.655 10.20899 379 489 17.3% 9.869 8.95559 442 542 20.1% 10.751 8.22221215 312 9.8% 11.241 7.86498 2195 2290 100.0% 13.701 6.45790 223 31210.2% 14.070 6.28943 34.2 128 1.6% 14.457 6.12183 595 692 27.1% 14.9145.93526 129 229 5.9% 15.402 5.74823 990 1093 45.1% 15.921 5.56201 114222 5.2% 16.286 5.43814 666 776 30.4% 17.046 5.19745 1012 1124 46.1%17.392 5.09476 473 585 21.5% 17.562 5.04590 505 616 23.0% 18.035 4.9145065.4 173 3.0% 18.371 4.82545 226 330 10.3% 19.280 4.60009 427 532 19.5%19.910 4.45589 1229 1339 56.0% 20.445 4.34050 406 517 18.5% 20.7524.27683 172 282 7.8% 20.915 4.24395 130 239 5.9% 21.235 4.18071 279 38712.7% 21.579 4.11474 799 904 36.4% 21.909 4.05363 38.7 139 1.8% 22.3833.96882 110 208 5.0% 22.665 3.91998 512 609 23.3% 23.147 3.83948 78.3174 3.6% 23.538 3.77658 417 509 19.0% 24.111 3.68807 64.8 149 3.0%24.670 3.60583 95.8 180 4.4% 25.533 3.48590 789 880 36.0% 26.170 3.4023944.5 137 2.0% 26.904 3.31131 518 614 23.6% 27.366 3.25638 114 213 5.2%27.575 3.23223 78.8 180 3.6% 28.167 3.16561 163 266 7.4% 28.282 3.15301146 250 6.7% 28.542 3.12484 146 250 6.7% 28.960 3.08070 32.3 137 1.5%29.228 3.05304 33.5 138 1.5% 29.485 3.02701 28.9 135 1.3% 30.066 2.96986155 265 7.0% 30.461 2.93226 104 215 4.8% 31.002 2.88223 189 299 8.6%31.440 2.84310 62.4 169 2.8% 32.056 2.78984 34.6 139 1.6% 32.362 2.7641690.1 195 4.1% 33.279 2.69008 99.6 202 4.5% 34.511 2.59683 59.1 157 2.7%36.261 2.47542 46.4 136 2.1% 37.084 2.42233 45.3 136 2.1% 37.386 2.4034833.8 123 1.5% 38.983 2.30860 46.3 126 2.1%

In certain embodiments, Form A is characterized by a DSC thermogramcomprising an endotherm peak value at about 185.0° C.-194.0° C. In someembodiments, the endotherm peak value is at a temperature ranging fromabout 186.0° C.-193.0° C., from about 187.0° C.-192.0° C., or from about188.0° C.-191.0° C. In some more specific embodiments, the endothermpeak value is at about 189.1° C.

In certain embodiments, Form A is characterized by a DSC thermogramcomprising an endotherm peak value at about 148.0° C.-155.0° C. In someembodiments, the endotherm peak value is at a temperature ranging fromabout 150.0° C.-154.0° C., from about 151.0° C.-153.0° C., or from about151.5° C.-152.5° C. as determined by differential scanning calorimetry.In some more specific embodiments, the endotherm peak value is at about152.1° C.

In certain embodiments, Form A is characterized by a DSC thermogramcomprising endotherm peak values at about 185.0° C.-194.0° C. and atabout 148.0° C.-155.0° C. In some embodiments, the endotherm peak valuesare at a temperature ranging from about 186.0° C.-193.0° C., from about187.0° C.-192.0° C., or from about 188.0° C.-191.0° C. and from about150.0° C.-154.0° C., from about 151.0° C.-153.0° C., or from about151.5° C.-152.5° C. In some more specific embodiments, the endothermpeak values are at about 189.1° C. and at about 152.1° C.

In certain embodiments, Form A is characterized by a DSC thermogramcomprising an endotherm peak in units ° C. at about 107.8. In certainembodiments, the DSC thermogram comprises an endotherm peak in units °C. at about 152.1. In certain embodiments, the DSC thermogram comprisesan endotherm peak in units ° C. at about 189.1. In certain embodiments,the DSC thermogram comprises endotherm peaks in units ° C. at about107.8, about 152.1, and about 189.1. In certain embodiments, the DSCthermogram is substantially identical to that of FIG. 64.

In certain embodiments, Form A is characterized by a TGA thermogramshowing weight loss of about 1.8% at 160° C. In certain embodiments, theTGA thermogram is substantially identical to that of FIG. 64.

In certain embodiments, a tartrate salt having a stoichiometry of about2:1 comprises Form A. In certain embodiments, a tartrate salt having astoichiometry of about 2:1 consists essentially of Form A. In certainembodiments, Form A is essentially pure. Physical and chemicalproperties of Form A are described in Example 26, Table 17.Toxicokinetic and toxicology profiles of Form A are described in Example27.

e. Forms C, E, F, G, H, and I

The tartrate salt of the compound of Structure (I) exists in othercrystalline forms, as summarized in Table 2.

TABLE 2 Crystalline forms of tartrate salts of Structure (I) SolventSlurry Slurry Moisture Crystal screen from screen from screen fromStability DSC sorption form Form A Form A Form D Stoichiometry by HPLCOnset (° C.) isotherm A + + + + + + Not formed 1:1.5 Stable 170.52hygroscopic 1:2  Stable 187.06 hygroscopic B + + 1:1.2 131.62hygroscopic C + Not formed 165.76 hygroscopic D Not formed + + 1:1 Stable 127.43 hygroscopic E + 102.33 F + + 61.06 G + 128.61 H + 139.36I + 118.60 + + +: Formed in three solvent systems or more + +: Formed ina few solvent systems +: Formed in one solvent systems or only at thehigher temperature

From polymorph screening studies, ten crystal forms, Form A (1:2), A′(1:1.5), B, C, D, E, F, G, H and I, were assigned by XRPD patterns asshown in FIG. 98. The thermal behavior (DSC/TGA charts) for thesecrystalline forms are shown in FIGS. 99A-99I.

The Slurry Screens and Solvent Screens described below were performedaccording to the methods of Example 51.

TABLE 3 Slurry Screening of Form A Room Temperature 50° C. CrystalCrystal Run Solvent Form Run Solvent Form 1-1 Methanol A 2-1 Methanol A1-2 Ethanol A 2-2 Ethanol A 1-3 2-Propanol A 2-3 2-Propanol A 1-42-Propanol- A 2-4 2-Propanol- A H₂O(5:1) H₂O(5:1) 1-5 H₂O B 2-5 H₂O —1-6 Methanol- A 2-6 Methanol- — H₂O(5:1) H₂O(5:1) 1-7 Ethanol- A 2-7Ethanol- A H₂O(5:1) H₂O(5:1) 1-8 Methanol- A 2-8 Methanol- A H₂O(10:1)H₂O(10:1) 1-9 Acetonitrile- — 2-9 Acetonitrile- — H₂O(1:1) H₂O(1:1) 1-10 Acetonitrile- A  2-10 Acetonitrile- A H₂O(10:1) H₂O(10:1)

TABLE 4 Solvent Screen of Form A′ Run Crystal Form Solvent 001 B H₂O 002— Methanol 003 A Ethanol 004 A 2-Propanol 005 A 2-Butanol 006 AChloroform 007 A Acetonitrile 008 A 1,2-Dimethoxyethane 009 ATetrahydrofuran 010 A Butyl methyl ether 011 A Cyclopentyl methyl ether012 A Ethyl acetate 013 A Propyl acetate 014 A Acetone 015 A Heptane 016A Chlorobenzene 017 A Toluene 018 A + C H₂O-Methanol(1:1) 019 —H₂O-Ethanol(1:1) 020 C H₂O-2-Propanol (1:1) 021 — H₂O-Acetonitrile(1:1)022 — H₂O-Acetone(1:1) 023 Amorphous H₂O-Methanol(1:10) 024 AmorphousH₂O-Ethanol(1:10) 025 Amorphous + A H₂O-2-Propanol (1:10) 026Amorphous + A H₂O-Acetonitrile(1:10) 027 Amorphous + A H₂O-Acetone(1:10)028 C H₂O-2-Propanol (1:5) 029 — H₂O-Acetonitrile(1:5) 030 —H₂O-Acetone(1:5)

TABLE 5 Slurry Screen of Form A′ Room Temperature 50° C. Crystal CrystalRun Solvent Form Run Solvent Form 1-1 Methanol A 2-1 Methanol A 1-2Ethanol A 2-2 Ethanol A 1-3 2-Propanol A 2-3 2-Propanol A 1-4 Acetone A2-4 Acetone A 1-5 Acetonitrile A 2-5 Acetonitrile A 1-6 2-Propanol- A2-6 2-Propanol- A H₂O(10:1) H₂O(10:1) 1-7 2-Propanol- A 2-7 2-Propanol-A H₂O(5:1) H₂O(5:1) 1-8 Methyl tert- A 2-8 Methyl tert- A butyl etherbutyl ether 1-9 2-Propanol- A 2-9 2-Propanol- — H₂O(1:1) H₂O(1:1)

TABLE 6 Slurry Screen of Form D Room Temperature 50° C. Crystal CrystalRun Solvent Form Run Solvent Form 1-1 Methanol E 2-1 Methanol F 1-2Ethanol D 2-2 Ethanol D 1-3 2-Propanol D 2-3 2-Propanol D 1-42-Propanol- F 2-4 2-Propanol- F H₂O(5:1) H₂O(5:1) 1-5 H₂O G 2-5 H₂O G1-6 Methanol- H 2-6 Methanol- — H₂O(5:1) H₂O(5:1) 1-7 Ethanol- F 2-7Ethanol- F H₂O(5:1) H₂O(5:1) 1-8 Ethanol- D + F 2-8 Ethanol- F H₂O(10:1)H₂O(10:1) 1-9 Acetonitrile- — 2-9 Acetonitrile- — H₂O(1:1) H₂O(1:1) 1-10 Acetonitrile- F  2-10 Acetonitrile- I H₂O(10:1) H₂O(10:1)

In the solvent screen shown in Table 4 (above), Form C was formed by therecrystallization of Form A′ with the mixture of H₂O and alcohol, suchas methanol and 2-propanol. About 10% weight loss and a broad endothermpeak was observed in the thermal analysis chart as shown in FIG. 99C.That suggested that Form C might be a solvate with alcohol and thealcohol was eliminated depending on the increase of temperature.

Form E was formed in the slurry screen from Form D only in methanol atroom temperature, as shown in Table 6. About 4% weight loss was observedin the thermal analysis chart as shown in FIG. 99E.

Form F was formed in the slurry screen from Form D in the mixture ofalcohol and H₂O at room temperature and 50° C. as shown in Table 6.About 6%-weight loss was observed in the thermal analysis chart as shownin FIG. 99F.

Form G was formed in the slurry screen from Form D in H₂O at roomtemperature and 50° C. as shown in Table 6. The thermal analysis chartwas provided in FIG. 99G.

Form H was formed in the slurry screen from Form D only in Methanol-H₂O(5:1) at room temperature, as shown in 6. The thermal analysis chart isprovided in FIG. 99H.

Form I was formed in the slurry screen from Form D only inacetonitrile-H₂O (10:1) at 50° C., as shown in Table 6. Thermal analysisdata is provided in FIG. 99I.

C. Pharmaceutical Compositions

In another aspect, the present disclosure provides pharmaceuticalcompositions comprising one or more compounds described herein, such asa pharmaceutically acceptable salt (e.g., a tartrate salt) of thecompound of structure (I), or a crystalline form thereof (e.g., Form A),or a compound of structure (IV), or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier or excipient. As usedherein, “a compound of structure (I)” is intended to refer to either thecompound per se (i.e., freebase), or a pharmaceutically acceptable saltsuch as a tartrate salt of the compound of structure (I), or acrystalline form thereof (e.g., Form A), unless it is specifiedotherwise. As used herein, “a compound of structure (IV)” is intended torefer to the compound per se, or an embodiment thereof as describedherein, including pharmaceutically acceptable salts thereof (e.g., atartrate salt).

In some embodiments, the pharmaceutical composition is formulated fororal administration. For example, in some embodiments, thepharmaceutical composition comprises an oral capsule. In otherembodiments, the pharmaceutical composition is formulated for injection.In some more specific embodiments, the carrier or excipient is selectedfrom the group consisting of cellulose, lactose, carboxymethylcelluloseand magnesium stearate.

In still more embodiments, the pharmaceutical compositions comprise acompound of structure (I), or a pharmaceutically acceptable salt thereof(e.g., tartrate salt), or a compound of structure (IV), and anadditional therapeutic agent (e.g., anticancer agent). Non-limitingexamples of such therapeutic agents are described herein below (e.g.,Ibrutinib or Alvocidib).

Suitable routes of administration include, but are not limited to, oral,intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary,transmucosal, transdermal, vaginal, otic, nasal, and topicaladministration. In addition, by way of example only, parenteral deliveryincludes intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, the pharmaceutically acceptable salt (e.g.,tartrate salt) of the compound of structure (I), or the compound ofstructure (IV), as described herein is administered in a local ratherthan systemic manner, for example, via injection directly into an organ,often in a depot preparation or sustained release formulation. Inspecific embodiments, long acting formulations are administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Furthermore, in other embodiments, the drug isdelivered in a targeted drug delivery system, for example, in a liposomecoated with organ-specific antibody. In such embodiments, the liposomesare targeted to and taken up selectively by the organ. In yet otherembodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), as described herein is provided in the form of a rapidrelease formulation, in the form of an extended release formulation, orin the form of an intermediate release formulation. In yet otherembodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), as described herein is administered topically.

The compound of structure (I) or pharmaceutically acceptable salt of thecompound of structure (I) (e.g., a tartrate salt), or the compound ofstructure (IV), according to certain embodiments is effective over awide dosage range. For example, in the treatment of adult humans,dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 5 to 100 mg, from20 to 100 mg, from 25 to 75 mg, from 1 to 50 mg per day, and from 5 to40 mg per day are examples of dosages that are used in some embodiments.An exemplary dosage is 10 to 30 mg per day. In various embodiments, thedosage is 3, 6, 9, 12, 16, 21, 28, 32, 42, or 50 mg per day. The exactdosage will depend upon the route of administration, the form in whichthe compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV), isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

In certain embodiments, the dosage of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt) isabout 1-37 mg/m² (e.g., 1-25 mg/m²) or about 1-75 mg (e.g., 1-50 mg)daily.

In certain embodiments, the daily dosage of the compound of structure(I) (e.g., freebase, or a pharmaceutically acceptable salt thereof suchas a tartrate salt), or the compound of structure (IV) can be about 20mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg,about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100mg.

In some embodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), is administered in a single dose. Typically, suchadministration will be by injection, e.g., intravenous injection, inorder to introduce the agent quickly. However, other routes are used asappropriate. A single dose of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), may also be used for treatment of an acutecondition.

In some embodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), is administered in multiple doses. In some embodiments,dosing is about once, twice, three times, four times, five times, sixtimes, or more than six times per day. In other embodiments, dosing isabout once a month, once every two weeks, once a week, or once everyother day. In another embodiment the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), and another agent (e.g., Ibrutinib orAlvocidib) are administered together about once per day to about 6 timesper day. In another embodiment the administration of the compound ofstructure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt), or the compound of structure (IV), and an agentcontinues for less than about 7 days. In yet another embodiment theadministration continues for more than about 6, 10, 14, 28 days, twomonths, six months, or one year. In some cases, continuous dosing isachieved and maintained as long as necessary.

Administration of the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), may continue as long as necessary. In some embodiments,the compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV), isadministered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In someembodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), is administered for less than 28, 14, 7, 6, 5, 4, 3, 2,or 1 day. In some embodiments, the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is administered chronically on an ongoingbasis, e.g., for the treatment of chronic effects. In some embodiments,the compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV), isadministered once daily for the first 21 out of a 28 day cycle.

In some embodiments, a compound of structure (I) or a pharmaceuticallyacceptable salt thereof is administered for 1, 2, 3, 4, 5, 6, or morecycles. In some embodiments, a compound of structure (I) or apharmaceutically acceptable salt thereof is administered in treatmentcycles until the subject presents progressive disease or no longertolerates treatment. In some embodiments, a compound of structure (I) ora pharmaceutically acceptable salt thereof is administered in treatmentcycles until the subject presents no detectable disease. In someembodiments, a compound of structure (I) or a pharmaceuticallyacceptable salt thereof is administered chronically on an ongoing basis,e.g., for the treatment of chronic effects.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintaindesired pharmacological effects. These plasma levels are referred to asminimal effective concentrations (MECs). Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. In someembodiments, methods of treatment comprise maintaining plasma levelsabove the MEC for 10-90% of the time. In some embodiments, plasma levelsare maintained between 30-90%. In some embodiments, plasma levels aremaintained between 50-90%. For example, in certain embodiments,effective amounts of a therapeutic agent may range from approximately2.5 mg/m2 to 1500 mg/m2 per day. Additional illustrative amounts rangefrom 0.2-1000 mg/qid, 2-500 mg/qid, and 20-250 mg/qid.

In some embodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or a compound ofstructure (IV), is administered in dosages. It is known in the art thatdue to intersubject variability in compound pharmacokinetics,individualization of dosing regimen is necessary for optimal therapy.Dosing for the compound of structure (I) or pharmaceutically acceptablesalt thereof (e.g., a tartrate salt), or a compound of structure (IV),may be found by routine experimentation in light of the instantdisclosure.

In some embodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or a compound ofstructure (IV), is formulated into pharmaceutical compositions. Inspecific embodiments, pharmaceutical compositions are formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Any pharmaceutically acceptable techniques,carriers, and excipients are used as suitable to formulate thepharmaceutical compositions described herein: Remington: The Science andPractice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack PublishingCompany, 1995); Hoover, John E., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins 1999).

In certain embodiments, the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or acompound of structure (IV), described are administered as pharmaceuticalcompositions in which the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or a compound ofstructure (IV), is mixed with other active ingredients, as incombination therapy. Encompassed herein are all combinations of activesset forth in the combination therapies section below and throughout thisdisclosure.

A pharmaceutical composition, as used herein, refers to a mixture of thecompound of structure (I) or pharmaceutically acceptable salt thereof(e.g., a tartrate salt), or a compound of structure (IV), with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Incertain embodiments, the pharmaceutical composition facilitatesadministration of the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), to a subject. In some embodiments, practicing themethods of treatment or use provided herein, therapeutically effectiveamounts of the compound of structure (I) or pharmaceutically acceptablesalt thereof (e.g., a tartrate salt), or the compound of structure (IV),provided herein are administered in a pharmaceutical composition to amammal having a disease, disorder or medical condition to be treated. Inspecific embodiments, the mammal is a human. In certain embodiments,therapeutically effective amounts vary depending on the severity of thedisease, the age and relative health of the subject, the potency of thecompound used and other factors. The compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), described herein is used singly or incombination with one or more therapeutic agents as components ofmixtures.

In some embodiments, the mammal (e.g., a human) is older than 18 yearsold. In some embodiments, the mammal (e.g., a human) has a lifeexpectancy of ≥3 months, is not pregnant or cannot become pregnant, haveacceptable liver function (e.g., bilirubin≤1.5× the upper limit ofnormal, or ≤3.0× the upper limit of normal for patients receivingimmunotherapy, aspartate aminotransferase, alanine aminotransferase andalkaline phosphatase≤2.5× the upper limit of normal), have acceptablerenal function (e.g., calculated creatine clearance≥30 mL/minute), haveacceptable hematologic status (e.g., granulocyte≥1500 cells/mm³,platelet count≥100,000 platelets/mm³ or hemoglobin≥9 g/dL), have noclinically significant abnormalities on urinalysis, have acceptablecoagulation status (e.g., prothrombin time within 1.5× normal limits oractivated partial thromboplastin time within 1.5× normal limits), or anycombination thereof.

In one embodiment, one or more of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is formulated in an aqueous solution. Inspecific embodiments, the aqueous solution is selected from, by way ofexample only, a physiologically compatible buffer, such as Hank'ssolution, Ringer's solution, or physiological saline buffer. In otherembodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), is formulated for transmucosal administration. Inspecific embodiments, transmucosal formulations include penetrants thatare appropriate to the barrier to be permeated. In still otherembodiments wherein the compounds described herein are formulated forother parenteral injections; appropriate formulations include aqueous ornon-aqueous solutions. In specific embodiments, such solutions includephysiologically compatible buffers and/or excipients.

In another embodiment, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), described herein is formulated for oral administration.Compounds described herein are formulated by combining the activecompounds with, e.g., pharmaceutically acceptable carriers orexcipients. In various embodiments, the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), described herein is formulated in oraldosage forms that include, by way of example only, tablets, powders,pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries,suspensions and the like.

In certain embodiments, pharmaceutical preparations for oral use areobtained by mixing one or more solid excipient with the compound ofstructure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt), or the compound of structure (IV), described herein,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are, in particular, fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;cellulose preparations such as: for example, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methylcellulose,microcrystalline cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP orpovidone) or calcium phosphate. In specific embodiments, disintegratingagents are optionally added. Disintegrating agents include, by way ofexample only, cross-linked croscarmellose sodium, polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

In one embodiment, dosage forms, such as dragee cores and tablets, areprovided with one or more suitable coating. In specific embodiments,concentrated sugar solutions are used for coating the dosage form. Thesugar solutions, optionally contain additional components, such as byway of example only, gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dyestuffs and/orpigments are also optionally added to the coatings for identificationpurposes. Additionally, the dyestuffs and/or pigments are optionallyutilized to characterize different combinations of active compounddoses.

In certain embodiments, therapeutically effective amounts of thecompound of structure (I) or pharmaceutically acceptable salt thereof(e.g., a tartrate salt), or the compound of structure (IV), describedherein are formulated into other oral dosage forms. Oral dosage formsinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In specific embodiments, push-fit capsules contain the activeingredients in admixture with one or more filler. Fillers include, byway of example only, lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In other embodiments, soft capsules contain the compound ofstructure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt), or the compound of structure (IV), that is dissolved orsuspended in a suitable liquid. Suitable liquids include, by way ofexample only, one or more fatty oil, liquid paraffin, or liquidpolyethylene glycol. In addition, stabilizers are optionally added.

In other embodiments, a therapeutically effective amount of the compoundof structure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt), or the compound of structure (IV), described herein isformulated for buccal or sublingual administration. Formulationssuitable for buccal or sublingual administration include, by way ofexample only, tablets, lozenges, or gels. In still other embodiments,the compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV),described herein are formulated for parental injection, includingformulations suitable for bolus injection or continuous infusion. Inspecific embodiments, formulations for injection are presented in unitdosage form (e.g., in ampoules) or in multi-dose containers.Preservatives are, optionally, added to the injection formulations. Instill other embodiments, the pharmaceutical compositions are formulatedin a form suitable for parenteral injection as sterile suspensions,solutions or emulsions in oily or aqueous vehicles. Parenteral injectionformulations optionally contain formulatory agents such as suspending,stabilizing and/or dispersing agents. In specific embodiments,pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form. Inadditional embodiments, suspensions of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), are prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles for use in thepharmaceutical compositions described herein include, by way of exampleonly, fatty oils such as sesame oil, or synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes. In certain specificembodiments, aqueous injection suspensions contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension containssuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, in other embodiments, the active ingredient is in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

In still other embodiments, the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is administered topically. The compoundsdescribed herein are formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical compositions optionally contain solubilizers,stabilizers, tonicity enhancing agents, buffers and preservatives.

In yet other embodiments, the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is formulated for transdermaladministration. In specific embodiments, transdermal formulations employtransdermal delivery devices and transdermal delivery patches and can belipophilic emulsions or buffered, aqueous solutions, dissolved and/ordispersed in a polymer or an adhesive. In various embodiments, suchpatches are constructed for continuous, pulsatile, or on demand deliveryof pharmaceutical agents. In additional embodiments, the transdermaldelivery of the compound of structure (I) or pharmaceutically acceptablesalt thereof (e.g., a tartrate salt), or the compound of structure (IV),is accomplished by means of iontophoretic patches and the like. Incertain embodiments, transdermal patches provide controlled delivery ofthe compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV). Inspecific embodiments, the rate of absorption is slowed by usingrate-controlling membranes or by trapping the compound of structure (I)or pharmaceutically acceptable salt thereof (e.g., a tartrate salt), orthe compound of structure (IV), within a polymer matrix or gel. Inalternative embodiments, absorption enhancers are used to increaseabsorption. Absorption enhancers or carriers include absorbablepharmaceutically acceptable solvents that assist passage through theskin. For example, in one embodiment, transdermal devices are in theform of a bandage comprising a backing member, a reservoir containingthe compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV),optionally with carriers, optionally a rate controlling barrier todeliver the compound of structure (I) or pharmaceutically acceptablesalt thereof (e.g., a tartrate salt), or the compound of structure (IV),to the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.

In other embodiments, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), is formulated for administration by inhalation. Variousforms suitable for administration by inhalation include, but are notlimited to, aerosols, mists or powders. Pharmaceutical compositions ofany of the compound of structure (I) or pharmaceutically acceptable saltthereof (e.g., a tartrate salt), or the compound of structure (IV), areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Inspecific embodiments, the dosage unit of a pressurized aerosol isdetermined by providing a valve to deliver a metered amount. In certainembodiments, capsules and cartridges of, such as, by way of exampleonly, gelatin for use in an inhaler or insufflator is formulatedcontaining a powder mix of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), and a suitable powder base such as lactoseor starch.

In still other embodiments, the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is formulated in rectal compositions such asenemas, rectal gels, rectal foams, rectal aerosols, suppositories, jellysuppositories, or retention enemas, containing conventional suppositorybases such as cocoa butter or other glycerides, as well as syntheticpolymers such as polyvinylpyrrolidone, PEG, and the like. In suppositoryforms of the compositions, a low-melting wax such as, but not limitedto, a mixture of fatty acid glycerides, optionally in combination withcocoa butter is first melted.

In certain embodiments, pharmaceutical compositions are formulated inany conventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Any pharmaceutically acceptable techniques,carriers, and excipients are optionally used as suitable. Pharmaceuticalcompositions comprising the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), are manufactured in a conventional manner,such as, by way of example only, by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or compression processes.

Additionally, the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), described herein encompass unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms of the compound of structure(I) or pharmaceutically acceptable salt thereof (e.g., a tartrate salt),or the compound of structure (IV), presented herein are also consideredto be disclosed herein. In addition, the pharmaceutical compositionsoptionally include other medicinal or pharmaceutical agents, carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressure,buffers, and/or other therapeutically valuable substances.

Methods for the preparation of compositions comprising the compound ofstructure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt), or the compound of structure (IV), described hereininclude formulating the compound of structure (I) or pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), or the compound ofstructure (IV), with one or more inert, pharmaceutically acceptableexcipients or carriers to form a solid, semi-solid or liquid. Solidcompositions include, but are not limited to, powders, tablets,dispersible granules, capsules, cachets, and suppositories. Liquidcompositions include solutions in which the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is dissolved, emulsions comprising acompound, or a solution containing liposomes, micelles, or nanoparticlescomprising the compound of structure (I) or pharmaceutically acceptablesalt thereof (e.g., a tartrate salt), or the compound of structure (IV),as disclosed herein. Semi-solid compositions include, but are notlimited to, gels, suspensions and creams. The form of the pharmaceuticalcompositions described herein include liquid solutions or suspensions,solid forms suitable for solution or suspension in a liquid prior touse, or as emulsions. These compositions also optionally contain minoramounts of nontoxic, auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, and so forth.

In some embodiments, the pharmaceutical composition comprising thecompound of structure (I) or pharmaceutically acceptable salt thereof(e.g., a tartrate salt), or the compound of structure (IV),illustratively takes the form of a liquid where the agents are presentin solution, in suspension or both. Typically when the composition isadministered as a solution or suspension a first portion of the agent ispresent in solution and a second portion of the agent is present inparticulate form, in suspension in a liquid matrix. In some embodiments,a liquid composition includes a gel formulation. In other embodiments,the liquid composition is aqueous.

In certain embodiments, useful aqueous suspensions contain one or morepolymers as suspending agents. Useful polymers include water-solublepolymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. Certain pharmaceutical compositionsdescribed herein comprise a mucoadhesive polymer, selected for examplefrom carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

Useful pharmaceutical compositions also, optionally, includesolubilizing agents to aid in the solubility of the compound ofstructure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt), or the compound of structure (IV). The term“solubilizing agent” generally includes agents that result in formationof a micellar solution or a true solution of the agent. Certainacceptable nonionic surfactants, for example polysorbate 80, are usefulas solubilizing agents, as can ophthalmically acceptable glycols,polyglycols, e.g., polyethylene glycol 400, and glycol ethers.

Furthermore, useful pharmaceutical compositions optionally include oneor more pH adjusting agents or buffering agents, including acids such asacetic, boric, citric, lactic, phosphoric and hydrochloric acids; basessuch as sodium hydroxide, sodium phosphate, sodium borate, sodiumcitrate, sodium acetate, sodium lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases and buffersare included in an amount required to maintain pH of the composition inan acceptable range.

Additionally, useful compositions also, optionally, include one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

Other useful pharmaceutical compositions optionally include one or morepreservatives to inhibit microbial activity. Suitable preservativesinclude mercury-containing substances such as merfen and thiomersal;stabilized chlorine dioxide; and quaternary ammonium compounds such asbenzalkonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride.

Still other useful compositions include one or more surfactants toenhance physical stability or for other purposes. Suitable nonionicsurfactants include polyoxyethylene fatty acid glycerides and vegetableoils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40.

Still other useful compositions include one or more antioxidants toenhance chemical stability where required. Suitable antioxidantsinclude, by way of example only, ascorbic acid and sodium metabisulfite.

In certain embodiments, aqueous suspension compositions are packaged insingle-dose non-reclosable containers. Alternatively, multiple-dosereclosable containers are used, in which case it is typical to include apreservative in the composition.

In alternative embodiments, other delivery systems for hydrophobicpharmaceutical compounds are employed. Liposomes and emulsions areexamples of delivery vehicles or carriers useful herein. In certainembodiments, organic solvents such as N-methylpyrrolidone are alsoemployed. In additional embodiments, the compounds described herein aredelivered using a sustained-release system, such as semipermeablematrices of solid hydrophobic polymers containing the therapeutic agent.Various sustained-release materials are useful herein. In someembodiments, sustained-release capsules release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein stabilization are employed.

In certain embodiments, the formulations described herein comprise oneor more antioxidants, metal chelating agents, thiol containing compoundsand/or other general stabilizing agents. Examples of such stabilizingagents, include, but are not limited to: (a) about 0.5% to about 2% w/vglycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% toabout 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e)about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/vpolysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h)arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1)pentosan polysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

In some embodiments, the concentration of the compound of structure (I)or pharmaceutically acceptable salt thereof (e.g., a tartrate salt), orthe compound of structure (IV), provided in the pharmaceuticalcompositions is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%,0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%,0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%,0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w,w/v or v/v.

In some embodiments, the concentration of the compound of structure (I)or pharmaceutically acceptable salt thereof (e.g., a tartrate salt), orthe compound of structure (IV), is in the range from approximately0.0001% to approximately 50%, approximately 0.001% to approximately 40%,approximately 0.01% to approximately 30%, approximately 0.02% toapproximately 29%, approximately 0.03% to approximately 28%,approximately 0.04% to approximately 27%, approximately 0.05% toapproximately 26%, approximately 0.06% to approximately 25%,approximately 0.07% to approximately 24%, approximately 0.08% toapproximately 23%, approximately 0.09% to approximately 22%,approximately 0.1% to approximately 21%, approximately 0.2% toapproximately 20%, approximately 0.3% to approximately 19%,approximately 0.4% to approximately 18%, approximately 0.5% toapproximately 17%, approximately 0.6% to approximately 16%,approximately 0.7% to approximately 15%, approximately 0.8% toapproximately 14%, approximately 0.9% to approximately 12%,approximately 1% to approximately 10% w/w, w/v or v/v.

In some embodiments, the concentration of the compound of structure (I)or pharmaceutically acceptable salt thereof (e.g., a tartrate salt), orthe compound of structure (IV), is in the range from approximately0.001% to approximately 10%, approximately 0.01% to approximately 5%,approximately 0.02% to approximately 4.5%, approximately 0.03% toapproximately 4%, approximately 0.04% to approximately 3.5%,approximately 0.05% to approximately 3%, approximately 0.06% toapproximately 2.5%, approximately 0.07% to approximately 2%,approximately 0.08% to approximately 1.5%, approximately 0.09% toapproximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v orv/v.

In some embodiments, the amount of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt), or thecompound of structure (IV), is equal to or less than 10 g, 9.5 g, 9.0 g,8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g,3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g,0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g,0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g,0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

Pharmaceutical compositions comprising a pharmaceutically acceptablesalt (e.g., tartrate salt) of the compound of structure (I), asdescribed above, may comprise a crystalline form as described herein(e.g., Form A). A polymorph of the present disclosure is typicallyformulated into pharmaceutical dosage forms to provide an easilycontrollable dosage of the drug and to give the subject an elegant andergonomic product. The dosage regimen for the polymorphs of the presentdisclosure will, of course, vary depending upon known factors, such asthe pharmacodynamic characteristics of the particular agent and its modeand route of administration; the species, age, sex, health, medicalcondition, and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment; the frequency of treatment;the route of administration, the renal and hepatic function of thesubject, and the effect desired. Polymorphs of this disclosure may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three, or four times daily.

In some embodiments, the pharmaceutical composition comprises thepolymorph in a concentration ranging from about 0.5 wt. % to about 5.0wt. %. In other specific embodiments, the pharmaceutical compositioncomprises the polymorph in a concentration ranging from about 1.8 wt. %to about 2.8 wt. %.

In some embodiments, the pharmaceutical composition comprises thepolymorph in a concentration ranging from about 10.0 wt. % to about 20.0wt. %. In other specific embodiments, the pharmaceutical compositioncomprises the polymorph in a concentration ranging from about 13.7 wt. %to about 15.7 wt. %.

In some embodiments, the pharmaceutical composition comprises thepolymorph in a concentration ranging from about 16.3 wt. % to about 36.3wt. %. In other specific embodiments, the pharmaceutical compositioncomprises the polymorph in a concentration ranging from about 21.3 wt. %to about 31.3 wt. %.

In more specific embodiments, the pharmaceutical composition comprisesabout 2.35 wt. % of the polymorph. In other specific embodiments, thepharmaceutical composition comprises about 14.7 wt. % of the polymorph.In still other specific embodiments, the pharmaceutical compositioncomprises about 26.3 wt. % of the polymorph.

In some more specific embodiments, the pharmaceutical compositioncomprises about 4 milligram (mg) of the polymorph. In other specificembodiments, the pharmaceutical composition comprises about 25 mg of thepolymorph. In some embodiments, the pharmaceutical composition comprisesabout 100 mg of the polymorph.

In some embodiments, the excipient is lactose (e.g., lactosemonohydrate), microcrystalline cellulose, croscarmellose sodium,magnesium stearate, or a combination thereof. In more specificembodiments, the excipient comprises lactose monohydrate. In someembodiments, the excipient comprises microcrystalline cellulose. In someembodiments, the excipient comprises croscarmellose sodium. In someembodiments, the excipient comprises magnesium stearate. In someembodiments, the excipient comprises microcrystalline cellulose, lactosemonohydrate, croscarmellose sodium, and magnesium stearate.

In some of the foregoing embodiments, the pharmaceutical compositioncomprises the excipient in a concentration ranging from about 95.0 wt. %to about 99.5 wt. %. In more specific embodiments, the pharmaceuticalcomposition comprises the excipient in a concentration ranging fromabout 97.2 wt. % to about 98.2 wt. %. In some embodiments, thepharmaceutical composition comprises about 97.65 wt. % of the excipient.

In some of the foregoing embodiments, the pharmaceutical compositioncomprises the excipient in a concentration ranging from about 80.0 wt. %to about 90.0 wt. %. In more specific embodiments, the pharmaceuticalcomposition comprises the excipient in a concentration ranging fromabout 84.3 wt. % to about 86.4 wt. %. In some embodiments, thepharmaceutical composition comprises about 85.3 wt. % of the excipient.

In some of the foregoing embodiments, the pharmaceutical compositioncomprises the excipient in a concentration ranging from about 63.7 wt. %to about 88.7 wt. %. In more specific embodiments, the pharmaceuticalcomposition comprises the excipient in a concentration ranging fromabout 68.7 wt. % to about 83.7 wt. %. In some embodiments, thepharmaceutical composition comprises about 73.7 wt. % of the excipient.

The polymorph of a tartaric acid salt of a compound of structure (I)according to certain embodiments is effective over a wide dosage range.For example, in the treatment of adult humans, dosages from 0.01 to 1000mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg perday are examples of dosages that are used in some embodiments. Anexemplary dosage is 10 to 30 mg per day. In various embodiments, thedosage is 3, 6, 9, 12, 16, 21, 28, 32, 42, or 50 mg per day. The exactdosage will depend upon the route of administration, the form in whichthe polymorph of a tartaric acid salt of a compound of structure (I) isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

Some particular embodiments provide a unit dose form comprising apharmaceutical composition as described herein. In various embodiments,the unit dose form is formulated for oral administration. In someembodiments, the unit dose form is a capsule. In some embodiments, theunit dose form is a tablet.

In some embodiments, the unit dose form comprises the polymorph in aconcentration ranging from about 0.5 wt. % to about 5.0 wt. %. In otherspecific embodiments, the unit dose form comprises the polymorph in aconcentration ranging from about 1.8 wt. % to about 2.8 wt. %.

In some embodiments, the unit dose form comprises the polymorph in aconcentration ranging from about 10.0 wt. % to about 20.0 wt. %. Inother specific embodiments, the unit dose form comprises the polymorphin a concentration ranging from about 13.7 wt. % to about 15.7 wt. %.

In some embodiments, the unit dose form comprises the polymorph in aconcentration ranging from about 16.3 wt. % to about 36.3 wt. %. Inother specific embodiments, the unit dose form comprises the polymorphin a concentration ranging from about 21.3 wt. % to about 31.3 wt. %.

In more specific embodiments, the unit dose form comprises about 2.35wt. % of the polymorph. In other specific embodiments, the unit doseform comprises about 14.7 wt. % of the polymorph. In still otherspecific embodiments, the unit dose form comprises about 26.3 wt. % ofthe polymorph.

In some more specific embodiments, the unit dose form comprises about 4milligram (mg) of the polymorph. In other specific embodiments, the unitdose form comprises about 25 mg of the polymorph. In some embodiments,the unit dose form comprises about 100 mg of the polymorph.

In some of the foregoing embodiments, the unit dose form comprises theexcipient in a concentration ranging from about 95.0 wt. % to about 99.5wt. %. In more specific embodiments, the unit dose form comprises theexcipient in a concentration ranging from about 97.2 wt. % to about 98.2wt. %. In other embodiments, the unit dose form comprises about 97.65wt. % of the excipient.

In some of the foregoing embodiments, the unit dose form comprises theexcipient in a concentration ranging from about 80.0 wt. % to about 90.0wt. %. In more specific embodiments, the unit dose form comprises theexcipient in a concentration ranging from about 84.3 wt. % to about 86.4wt. %. In other embodiments, the unit dose form comprises about 85.3 wt.% of the excipient.

In some of the foregoing embodiments, the unit dose form comprises theexcipient in a concentration ranging from about 63.7 wt. % to about 88.7wt. %. In more specific embodiments, the unit dose form comprises theexcipient in a concentration ranging from about 68.7 wt. % to about 83.7wt. %. In other embodiments, the unit dose form comprises about 73.7 wt.% of the excipient.

The pharmaceutical composition or combination of the present disclosurecan be in unit dosage of about 1-1000 mg of active ingredient(s) for asubject of about 50-70 kg, or about 1-500 mg or about 1-250 mg or about1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredients.The therapeutically effective dosage of a compound (e.g., a polymorph),the pharmaceutical composition, or the combinations thereof, isdependent on the species of the subject, the body weight, age andindividual condition, the disorder or disease or the severity thereofbeing treated. A physician, clinician or veterinarian of ordinary skillcan readily determine the therapeutically effective amount of each ofthe active ingredients necessary to prevent, treat or inhibit theprogress of the disorder or disease.

D. Therapeutic Uses and Methods of Treatment

1. Methods of Treating Cancer

Certain embodiments provide a method for treatment of cancer, the methodcomprising administering an effective amount of a pharmaceuticalcomposition comprising a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)as described herein to a subject in need thereof. In some embodiments,the methods comprising administering an effective amount of apharmaceutical composition comprising a compound of structure (I) to asubject in need thereof. In some different embodiments, the methodsdescribed herein comprise administering an effective amount of apharmaceutical composition comprising a pharmaceutically acceptable saltof a compound of structure (I) (e.g., a tartrate salt) to a subject inneed thereof. In some more specific embodiments, the cancer is ahematological cancer. In some embodiments, the cancer is a chroniclymphocytic leukemia.

In some specific embodiments, a method for treatment of cancer isprovided, wherein the method comprises administering a compound ofstructure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., tartrate salt), or a pharmaceutical compositioncomprising the same, to a subject in need thereof. In some more specificembodiments, the cancer is a hematological cancer, and the methodscomprise administering a pharmaceutically acceptable salt of a compoundof structure (I) (e.g., tartrate salt) to the subject. In someembodiments, the cancer is a chronic lymphocytic leukemia.

In some embodiments, a method for treatment of cancer is provided, themethod comprising administering an effective amount of any of theforegoing pharmaceutical compositions comprising a compound of structure(I), or pharmaceutically acceptable salt of a compound of structure (I),for example a tartrate salt of a compound of structure (I), to a subjectin need thereof. In some embodiments, the cancer is mediated by an AXLkinase. In some embodiments, the cancer is breast cancer (e.g., triplenegative breast cancer), pancreatic cancer, renal carcinoma, coloncancer, thyroid carcinoma, colorectal cancer (e.g., BRAF, KRAS, orNRAS-mutated colorectal carcinoma), ovarian cancer, melanoma (e.g.,BRAF-mutated melanoma) or lung cancer (e.g., EGFR+ non-small cell lungcancer). In certain embodiments, the cancer is selected from the groupconsisting of chronic lymphocytic leukemia (CLL), breast cancer, andcolon cancer. In particular embodiments, the cancer is an advanced solidtumor, triple negative breast cancer, EGFR+ non-small cell lung cancer,colorectal carcinoma, recurrent ovarian carcinoma, or BRAF-mutatedmelanoma.

In some embodiments, the cancer is an advanced solid tumor (e.g., anadvanced solid tumor that has progressed despite immunotherapy). In someembodiments is provided, a method for treating an advanced solid tumorcomprising administering an effective amount a compound of structure (I)or a pharmaceutically acceptable salt thereof (e.g., a tartrate salt).In some embodiments the advanced solid tumor is an advanced metastaticor progressive solid tumor. In some embodiments, the cancer (e.g., atumor) has progressed despite immunotherapy.

One embodiment provides a method for modulating a tumor immune responsein a subject in need thereof, wherein the method comprises administeringa compound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt), or a pharmaceuticalcomposition comprising the same, to the subject in need thereof therebymodulating a tumor immune response. In some embodiments, the immuneresponse is increased. In some embodiments, the tumor is a solid tumor.In some embodiments, the modulating comprises inhibiting TAM kinaseactivity. In some embodiments, the modulating comprises increasingactivated dendritic cells and/or increasing tumor infiltration bydendritic cells. In some embodiments, the modulating comprises reducingneutrophils, regulator T-cells or combinations thereof. In someembodiments, the modulating comprises reducing immunosuppressivecytokines (e.g., IL-6, G-CSF) in the tumor microenvironment. In someembodiments, the method further comprises administering atherapeutically effective amount of an additional agent, for example, animmune checkpoint inhibitor described herein or known in the art.

One embodiment provides a method for modulating an immune response in asubject in need thereof, wherein the method comprises administering acompound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt), or a pharmaceuticalcomposition comprising the same, to the subject in need thereof. In someembodiments, modulating an immune response includes modulating of immunecell populations (e.g., neutrophils, regulatory T-cells, and dendriticcells in a tumor).

One embodiment provides a method for enhancing host immunity in asubject in need thereof, wherein the method comprises administering acompound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt), or a pharmaceuticalcomposition comprising the same, to the subject in need thereof.

One embodiment provides a method for suppressing a mesenchymal phenotypein a subject in need thereof, wherein the method comprises administeringa compound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt), or a pharmaceuticalcomposition comprising the same, to the subject in need thereof.Suppressing a mesenchymal phenotype may include reversing a mesenchymalphenotype. Suppressing (e.g., reversing a mesenchymal phenotype) may beuseful, for example, for treating a wide variety of cancers linked to amesenchymal phenotype, such as metastatic colorectal cancer. Amesenchymal phenotype may be evidenced by the expression of mesenchymalmarkers, such as Snail and Slug. In particular embodiments of the methodfor suppressing a mesenchymal phenotype, a compound of structure (I) isadministered to a subject with metastatic colorectal cancer. Inparticular embodiments of the method for suppressing a mesenchymalphenotype, a compound of structure (I) is administered to a subject withnon-metastatic colorectal cancer, thereby preventing or reducing thelikelihood of metastasis of the colorectal cancer.

One embodiment provides a method for creating a tumor microenvironmentamendable to an immune response in a subject in need thereof, whereinthe method comprises administering a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt), or a pharmaceutical composition comprising the same, tothe subject in need thereof. In certain embodiments, creating a tumormicroenvironment amenable to an immune response includes reducing locallevels of immunosuppressive cytokines and chemokines, such as IL-6 andG-CSF. In certain embodiments, creating a tumor microenvironmentamenable to an immune response includes increased infiltration andactivation of dendritic cells (DCs) in the tumor.

One embodiment provides a method for treating cancer comprisingadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) incombination with an immunotherapy. Another embodiment provides a methodfor treating cancer comprising administering an effective amount acompound of structure (I) or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt) in combination with a tyrosine kinase inhibitor.

In embodiments, the cancer is breast cancer. Breast cancer iscategorized into four major molecular subtypes: (a) luminal A, which ishormone receptor positive (estrogen receptor positive and/orprogesterone receptor positive), human epidermal growth factor receptor2 (HER2) negative, and has low expression of Ki-67 (a cellular markerfor proliferation); (b) luminal B, which is hormone receptor positive,may be HER2 positive or negative, and has high expression of Ki-67; (c)HER2-enriched, which is hormone receptor negative and HER2 positive; and(d) triple negative, which is hormone receptor negative and HER2negative.

In some embodiments, the breast cancer is hormone receptor positive.Hormone receptor positive breast cancer refers to breast cancer that ispositive for expression of estrogen receptor and/or progesteronereceptor. In some embodiments, the breast cancer is luminal A or luminalB breast cancer. In particular embodiments, a method for treatinghormone receptor positive breast cancer (e.g., luminal A or luminal Bbreast cancer) comprises administering an effective amount a compound ofstructure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) and administering an effective amount of a hormonetherapy. Hormone therapy for breast cancer can refer to a therapy thatreduces estrogen binding to estrogen receptor, and/or reducesprogesterone binding to progesterone receptor. In particularembodiments, the hormone therapy comprises tamoxifen, toremifene,fulvestrant, or an aromatase inhibitor. Examples of aromatase inhibitorsused to treat hormone receptor positive breast cancer includeanastrozole, exemestane, and letrozole.

In some embodiments, the breast cancer is HER2 positive. HER2 positivebreast cancer is a breast cancer that tests positive for expression ofHER2, such as luminal B or HER2 enriched breast cancer. In someembodiments, a method for treating HER2 positive breast cancer comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of HER2 inhibitor. In someembodiments, the HER2 inhibitor is selected from trastuzumab,pertuzumab, trastuzumab emtansine (i.e., TDM1), lapatinib, andneratinib. In particular embodiments, the HER2 inhibitor is trastuzumabor trastuzumab emtansine.

In some embodiments, the breast cancer is triple negative breast cancer.Triple negative breast cancer is the most aggressive molecular subtypeof breast cancer, and is not responsive to hormone therapy regimens orHER2 inhibitors.

Breast cancer can also be categorized as inflammatory breast cancer ornon-inflammatory breast cancer. Inflammatory breast cancer is a rare,highly aggressive form of breast cancer, which is diagnosed based onclinical presentation with inflammatory-like symptoms in the breastincluding erythema, oedema, tenderness, warmth, and/or skin dimpling.Inflammatory breast cancer may present with the any of the molecularsubtypes of breast cancer (i.e., may be hormone receptor positive and/orHER2 positive, or may be triple negative).

In some embodiments, the cancer is inflammatory breast cancer.Inflammatory breast cancer may be hormone receptor positive and/or HER2positive, or triple negative. Inflammatory breast cancer may be, incertain cases, responsive to hormone therapy and or HER2 inhibitors,depending on the molecular subtype. Given that inflammatory breastcancer is highly aggressive, treatment often includes radical mastectomyin combination with chemotherapy and/or radiation.

In embodiments, methods for treating breast cancer (e.g., triplenegative breast cancer or inflammatory breast cancer) compriseadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a further therapy to a subject inneed thereof. The further therapy may be, for example, radiation,surgical (e.g., mastectomy), or a therapeutic agent (e.g., achemotherapeutic agent. In particular embodiments, the further therapyis at least one of: an immune checkpoint inhibitor, a PARP inhibitor, aWEE1 inhibitor, an EGFR targeting agent, a CDK4/6 inhibitor, a PI3Kinhibitor, a TORC½ inhibitor, an anthracycline, or a taxane.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of an immune checkpoint inhibitor to asubject in need thereof. In particular embodiments, the immunecheckpoint inhibitor is a PD-L1 inhibitor, a PD-1 inhibitor, a CTLA-4inhibitor, a LAG-3 inhibitor, a Tim-3 inhibitor, or a combinationthereof.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a PARP inhibitor to a subject inneed thereof. In some embodiments, the PARP inhibitor is selected fromolaparib, niraparib, talazoparib, and velaparib. In particularembodiments, the PARP inhibitor is olaparib.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a WEE1 inhibitor to a subject inneed thereof. WEE1 is a nuclear kinase that regulates cell cycleprogression, and inhibition of WEE1 has anti-cancer effects and maysensitive cancer cells to other treatments, such as cisplatin orradiation. In particular embodiments, the WEE1 inhibitor comprisesAZD1775.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of an EGFR targeting agent to asubject in need thereof. In particular embodiments, the EGFR-targetingagent is lapatinib, panitumumab, cetuximab or erlotinib, gefetinib, or acombination thereof.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a CDK4 or CDK6 inhibitor to asubject in need thereof. In some embodiments, the CDK4 or CDK6 inhibitoris palbociclib, abemaciclib, ribociclib, or a combination thereof. Inparticular embodiments, the CDK4 or CDK6 inhibitor is palbociclib.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a PI3K inhibitor to a subject inneed thereof. The PI3K-AKT-mTOR pathway is commonly activated in avariety of cancers and contributes to cancer cell survival inproliferation. In some embodiments, the PI3K inhibitor is AZD8186,GDC-0941, GDC-0980, idelalisib (CAL-101), alpelisib (BYL719), buparlisib(BKM120), or a combination thereof. In particular embodiments, the PI3Kinhibitor is AZD8186.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a TORC½ inhibitor to a subject inneed thereof. In some embodiments, the TORC½ inhibitor is AZD2014(vistusertib), TAK228 (INK128, MLN0128), or both.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of an anthracycline to a subject inneed thereof. “Anthracycline” refers to a chemical class of compoundswith intercalating activity, and include daunorubicin, doxorubicin,epirubicin, idarubicin, nemorubicin, pixantrone, sabarubicin, andvalrubicin. In particular embodiments, the anthracycline is doxorubicin,Daunorubicin, or both.

In certain embodiments, a method for treating breast cancer (e.g.,triple negative breast cancer or inflammatory breast cancer) comprisesadministering an effective amount a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering an effective amount of a taxane to a subject in needthereof. Taxanes used to treat cancer are di-terpene molecules thatinhibit microtubule formation. In some embodiments, the taxane comprisespaclitaxel, docetaxel, or both.

In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is renal carcinoma. In some embodiments, thecancer is colon cancer. In some embodiments, the cancer is thyroidcancer. In some embodiments, the cancer is lung cancer.

In some embodiments, the cancer is non-small cell lung cancer. Incertain embodiments, a method for treating non-small cell lung cancer(e.g., EGRF+ non-small cell lung cancer) comprises administering aneffective amount a compound of structure (I) or a pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt). In some embodiments,the compound of structure (I) or a pharmaceutically acceptable saltthereof (e.g., a tartrate salt) is administered in combination with anEGRF inhibitor, an immunotherapy and/or a tyrosine kinase inhibitor(e.g., CDK9 inhibitor). In some embodiments, the non-small cell lungcancer comprises an immunotherapy-resistant tumor. In some embodiments,the non-small cell lung cancer (e.g., EGRF+ non-small cell lung cancer)has progressed after administering≤2 lines of tyrosine kinase inhibitors(e.g., oral tyrosine kinase inhibitors).

In some embodiments, the cancer is colorectal cancer. In certainembodiments, the colorectal cancer includes tumor(s) that expressmutated BRAF, KRAS or NRAS. In certain embodiments, a method fortreating colorectal cancer comprising administering an effective amounta compound of structure (I) or a pharmaceutically acceptable saltthereof (e.g., a tartrate salt) in combination with an EGFR inhibitor.In some embodiments, the method comprises administering an immunecheckpoint inhibitor (e.g., PD-L1-, PD-1-, CTLA-4-, LAG-3- orTim-3-targeted agents). In some specific embodiments, the colorectalcancer is BRAF-, KRAS- or NRAS-mutated colorectal carcinoma. Inparticular embodiments, the colorectal cancer is KRAS-mutated colorectalcarcinoma. In some embodiments, the administration is after standardtherapies have been administered.

In some embodiments, the cancer is ovarian cancer. In some embodiments,a method for treating ovarian cancer comprising administering aneffective amount a compound of structure (I) or a pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt). Some embodiments theovarian cancer is persistent or recurrent. In some specific embodiments,the ovarian cancer is platinum refractory/resistant. In someembodiments, the method for treating ovarian cancer further comprisesadministering a platinum compound. In some embodiments, theadministering is following administration of any number of lines ofprior therapies.

In some embodiments, the cancer is melanoma (e.g., BRAF-mutatedmelanoma). In certain embodiments, a method for treating melanoma (e.g.,BRAF-mutated melanoma) comprising administering an effective amount acompound of structure (I) or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt). In some embodiments, the method furthercomprises administrating the compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) incombination with an immunotherapy or a combination BRAF and MEKinhibitor. In some embodiments, the melanoma (e.g., BRAF-mutatedmelanoma) has not responded to immunotherapy or a combination of aBRAF/MEK inhibitor.

One particular embodiment provides a method of treating a disease, themethod comprising administering an effective amount of the compound ofstructure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) or a pharmaceutical composition comprising the compoundof structure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) to a subject in need thereof, wherein the disease isselected from the group consisting of an advanced solid tumor, EGFR+non-small cell lung cancer, colorectal carcinoma, recurrent ovariancarcinoma, and BRAF-mutated melanoma.

In another embodiment the disclosure provides a method for inhibitingtumor metastasis, the method comprising administering an effectiveamount a compound of structure (I) or a pharmaceutically acceptable saltthereof (e.g., a tartrate salt) or a pharmaceutical composition ofcomprising a compound of structure (I) or pharmaceutically acceptablesalt of a compound of structure (I), for example a tartrate salt of acompound of structure (I) and a pharmaceutically acceptable carrier to asubject in need thereof.

One embodiment provides a method of treating a disease, the methodcomprising administering an effective amount of the compound ofstructure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) or a pharmaceutical composition comprising the compoundof structure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) to a subject in need thereof, wherein the disease iscolorectal cancer, for example, colorectal carcinoma. In someembodiments, the colorectal cancer is resistant to EGFR-targetedtherapies.

Hematologic malignancies that can be treated with a compound ofstructure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) include, but are not limited to leukemias and lymphomas.For example, the presently disclosed compounds and compositions can beused for treatment of diseases such as Acute lymphoblastic leukemia(AFF), Acute myelogenous leukemia (AMF), Chronic lymphocytic leukemia(CFF), small lymphocytic lymphoma (SEE), Chronic myelogenous leukemia(CMF), Acute monocytic leukemia (AMoF) and/or other leukemias. In otherembodiments, the compound of structure (I) or a pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt) is useful for treatmentof lymphomas such as all subtypes of Hodgkin's lymphoma or non-Hodgkin'slymphoma. In specific embodiments, the presently disclosed compounds andcompositions can be used for treatment of CFF and/or SLL. In someparticular embodiments, the presently disclosed compounds andcompositions can be used for treatment of CFF. In some particularembodiments, the presently disclosed compounds and compositions can beused for treatment of SLL.

A wide variety of cancers, including solid tumors and leukemias (e.g.,acute myeloid leukemia and chronic lymphocytic leukemia) are amenable tothe methods disclosed herein. Types of cancer that may be treated invarious embodiments include, but are not limited to: adenocarcinoma ofthe breast, prostate, and colon; all forms of bronchogenic carcinoma ofthe lung; myeloid; melanoma; hepatoma; neuroblastoma; papilloma;apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoidheart disease; and carcinoma (e.g., Walker, basal cell, basosquamous,Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous,non-small cell lung, oat cell, papillary, scirrhous, bronchiolar,bronchogenic, squamous cell, and transitional cell); histiocyticdisorders; leukemia; histiocytosis malignant; Hodgkin's disease;immunoproliferative small; plasmacytoma; reticuloendotheliosis;melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma;fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma;mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma;craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma;myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma;trophoblastic tumor; adenoma; cholangioma; cholesteatoma; cyclindroma;cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma;hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma;sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma;myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma;ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma;neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma;paraganglioma; paraganglioma non-chromaffin; angiokeratoma;angiolymphoid hyperplasia with eosinophilia; angioma sclerosing;angiomatosis; glomangioma; hemangioendothelioma; hemangioma;hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma;lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma;cystosarcoma phyllodes; hemangiosarcoma; leiomyosarcoma; leukosarcoma;liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovariancarcinoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; andcervical dysplasia.

In a still further aspect, the cancer is selected from cancers of thebrain, genitourinary tract, endocrine system, gastrointestinal tract,blood, rectum, kidney, lymphatic system, stomach, and skin.

In some embodiments, the cancer is selected from cancer in adolescents,adrenocortical carcinoma childhood, AIDS-related cancers (e.g., Lymphomaand Kaposi's Sarcoma), anal cancer, appendix cancer, astrocytomas,atypical teratoid, basal cell carcinoma, bile duct cancer, bladdercancer, bone cancer, brain stem glioma, brain tumor, breast cancer,bronchial tumors, Burkitt lymphoma, carcinoid tumor, atypical teratoid,embryonal tumors, germ cell tumor, primary lymphoma, cancer inadolescents, cervical cancer, childhood cancers, chordoma, cardiactumors, chronic myeloproliferative disorders, colon cancer, colorectalcancer (e.g., colorectal cancer that is resistant to EGFR-targetedtherapies), craniopharyngioma, cutaneous T-cell lymphoma, extrahepaticductal carcinoma in situ (DCIS), embryonal tumors, CNS cancer,endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor,extragonadal germ cell tumor, eye cancer, fibrous histiocytoma of bone,gall bladder cancer, gastric cancer, gastrointestinal stromal tumors(GIST), gastrointestinal carcinoid tumor, germ cell tumor, gestationaltrophoblastic tumor, hairy cell leukemia, head and neck cancer, heartcancer, liver cancer, hypopharyngeal cancer, intraocular melanoma, isletcell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngealcancer, lip and oral cavity cancer, liver cancer, lobular carcinoma insitu (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer withoccult primary, midline tract carcinoma, mouth cancer, multipleendocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm,mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative neoplasms, such as myelofibrosis,multiple myeloma, merkel cell carcinoma, malignant mesothelioma,malignant fibrous histiocytoma of bone and osteosarcoma, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oralcancer, lip and oral cavity cancer, oropharyngeal cancer, ovariancancer, pancreatic cancer, papillomatosis, paraganglioma, paranasalsinus and nasal cavity cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pleuropulmonary blastoma, primary central nervoussystem (CNS) lymphoma, prostate cancer, rectal cancer, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skincancer, stomach (gastric) cancer, small cell lung cancer, smallintestine cancer, soft tissue sarcoma, T-Cell lymphoma, testicularcancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter, trophoblastictumor, unusual cancers of childhood, urethral cancer, uterine sarcoma,vaginal cancer, vulvar cancer, Viral-Induced cancer, acoustic neuroma,glioma, meningioma, pituitary adenoma, schwannoma, CNS lymphoma,primitive neuroectodermal tumor, craniopharyngioma, chordoma,medulloblastoma, cerebral neuroblastoma, central neurocytoma,pineocytoma, pineoblastoma, atypical teratoid rhabdoid tumor,chondrosarcoma, choroid plexus carcinoma, choroid plexus papilloma,craniopharyngioma, dysembryoplastic neuroepithelial tumor,gangliocytoma, germinoma, hemangioblastoma, hemangiopercytoma,metastatic brain tumor cell, and glioma (e.g., glioblastoma multiforme,ependymoma, astrocytoma, oligodendroglioma, oligoastrocytoma, juvenilepilocytic astrocytoma, subependymal giant cell astrocytoma,ganglioglioma, subependymoma, pleomorphic xanthoastrocytom, anaplasticastrocytoma, glioblastoma multiforme, brain stem glioma,oligodendroglioma, ependymoma, oligoastrocytoma, cerebellar astrocytoma,desmoplastic infantile astrocytoma, subependymal giant cell astrocytoma,diffuse astrocytoma, mixed glioma, optic glioma, gliomatosis cerebri,paraganglioma, or ganglioglioma cell).

Embodiments of the invention also include a method of treating ahyperproliferative disorder in a mammal that comprises administering tosaid mammal a therapeutically effective amount of a compound ofstructure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt). In some embodiments, the methodrelates to the treatment of a non-cancerous hyperproliferative disordersuch as benign hyperplasia of the skin (e.g., psoriasis), restenosis, orprostate (e.g., benign prostatic hypertrophy (BPH)). In some embodimentsthe method relates to use of a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt) for treatment of fibrosis including, but not limited topulmonary fibrosis (e.g., idiopathic pulmonary fibrosis) and liverfibrosis.

In some embodiments, the invention provides methods of inhibiting kinaseactivity in a cell by contacting said cell with an amount of a compoundof structure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt) sufficient to inhibit the activityof AXL kinase. In some embodiments, the invention provides methods ofinhibiting AXL kinase activity in a tissue by contacting said tissuewith an amount of a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)sufficient to inhibit the activity of the AXL kinase in said tissue. Insome embodiments, the invention provides methods of inhibiting AXLkinase activity in an organism by contacting said organism with anamount of a compound of structure (I) or a pharmaceutically acceptablesalt of a compound of structure (I) (e.g., a tartrate salt) sufficientto inhibit the activity of the AXL kinase in said organism. In someembodiments, the invention provides methods of inhibiting AXL kinaseactivity in an animal by contacting said animal with an amount of acompound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt) sufficient to inhibitthe activity of the AXL kinase in said animal. In some embodiments, theinvention provides methods of inhibiting AXL kinase activity in a mammalby contacting said mammal with an amount of a pharmaceuticallyacceptable salt of a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)sufficient to inhibit the activity of AXL kinase in said mammal. In someembodiments, the invention provides methods of inhibiting AXL kinaseactivity in a human by contacting said human with an amount of acompound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt) sufficient to inhibitthe activity of AXL kinase in said human. In other embodiments, thepresent invention provides methods of treating a disease mediated by AXLkinase activity in a subject in need of such treatment.

In some specific embodiments, the present disclosure provides a methodof treating chronic lymphocytic leukemia (CLL) by administering an AXLkinase inhibitor, such as a compound of structure (I), or apharmaceutically acceptable salt thereof (e.g., tartrate salt), to asubject in need thereof. In some embodiments, methods of the disclosuretreat CLL and small lymphocytic lymphoma (SLL). In some embodiments, thesubject has been previously treated for CLL, yet still displays symptomsof detectable minimal residual disease (MRD). Accordingly, oneembodiment provides a method for treating CLL in a subject, comprisingadministering an effective amount of an AXL kinase inhibitor to thesubject, wherein the subject has received a prior treatment regimen forCLL, and the subject was refractory after the prior treatment regimen,the subject has relapsed CLL after a response to the prior treatmentregimen, or the subject has detectable minimal residual disease (MRD).

According to the revised IWCLL guidelines a subject diagnosed with CLLis considered to be in “complete remission” if peripheral blood(circulating) lymphocyte counts are normal, absence of significantlymphadenopathy (e.g., lymph nodes>1.5 cm in diameter) by physicalexamination, normal liver size and spleen size<13 cm, absence ofconstitutional symptoms, platelet count greater than or equal to100,000/μL, hemoglobin greater than or equal to 11.0 g/dL (untransfusedand without erythropoietin), and normocellular bone marrow (no CLLcells, no B-lymphoid nodules). A subject diagnosed with CLL meets thecriteria of “partial remission” if peripheral blood (circulating)lymphocyte counts decrease≥50% from baseline, lymph nodes decrease≥50%from baseline, liver and spleen sizes decrease≥50% from baseline,platelet count greater than or equal to 100,000/μL or increase≥50% frombaseline, hemoglobin greater than or equal to 11.0 g/dL or increase≥50%from baseline, yet still has the presence of CLL cell or B-lymphoidnodules in the bone marrow. For a “partial remission” at least oneparameter of the first three parameters needs to be met and oneparameter of the second three parameters needs to be met. The term“relapse” refers to disease progression in a patient who has previouslyachieved the above criteria of a CR or PR for a period of six or moremonths. In particular embodiments, the subject was refractory after theprior treatment regimen. In particular embodiments, the subject wasrelapsed CLL after a response to the prior treatment regimen. Inparticular embodiments, the subject has detectable minimal residualdisease (MRD).

In some embodiments, the subject has been previously treated for CLLand/or SLL. In some embodiments, a subject is intolerant to the priortreatment regimen. As used herein “intolerant” means that the subject isunable or unwilling to tolerate the adverse effects of an effectiveamount of therapeutic agent.

Detectable MRD refers to a disease state in which the subject has atleast 1 CLL cell per 10,000 leukocytes in a sample of peripheral bloodor bone marrow. Percent MRD refers to the percent of CLL cells relativeto leukocytes in a sample of peripheral blood or bone marrow. Forexample, a subject having 1 CLL cell per 10,000 leukocytes is classifiedas having 0.01% MRD. MRD can be determined from peripheral blood and/orbone marrow using techniques known in the art, for exampleimmunophenotyping (e.g., flow cytometry) or molecular based assays(e.g., polymerase chain reaction and next-generation sequencing), asdisclosed in JAMA Oncology, March 2018, Volume 4, Number 3, P. 394-400,and Blood, March 2018, blood-2017-09-806398, which are herebyincorporated by reference.

Exemplary AXL kinase inhibitors useful in treating CLL and/or SLL, suchas CLL with detectable MRD, and other embodiments of the invention areknown in the art, for example as disclosed in WO 2012/135800 and WO2008/128072, the full disclosures of which are hereby incorporated byreference in their entirety. Exemplary AXL kinase inhibitors includecompounds having the following structure (I′):

or a pharmaceutically acceptable salt, hydrate, solvate, or polymorphthereof, wherein:

L¹ is selected from O and NR⁵, wherein R⁵ is selected from hydrogen andC1-C6 alkyl;

L² is selected from CH₂ and NCH₃, provided that L² is CH₂ when Y is N;

Y is selected from CH and N;

Z is selected from O, NR⁶ and CH₂, wherein R⁶ is selected from hydrogenand CH₃;

each of R^(1a) and R^(1b) is independently selected from hydrogen,halogen, OH, CN, SO₂CH₃, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy,C1-C6 polyhaloalkoxy, C1-C6 haloalkyl, C1-C6 polyhaloalkyl, C1-C6cyanoalkyl, and NH(C═O)R⁷, R⁷ is selected from hydrogen and C1-C6 alkyl;

R² is selected from hydrogen, C1-C6 alkyl, SO₂R⁸, and (C═O)R⁸, whereinR⁸ is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6heterocycloalkyl, and NR¹⁰R¹¹, wherein: a) R¹⁰ is selected fromhydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; and R¹¹, when present, isselected from hydrogen and C1-C6 alkyl; or b) R¹⁰ and R¹¹ are covalentlybonded and, together with the intermediate nitrogen, comprise anoptionally substituted 3-7 membered heterocycloalkyl ring;

R³ is selected from hydrogen, halogen, OH, CN, C1-C6 alkyl, C1-C6haloalkyl, C1-C6 polyalkyl, C3-C6 cycloalkyl, C3-C6 haloalkyl, C3-C6polyhaloalkyl, and C3-C6 heterocycloalkyl;

R⁴ is selected from hydrogen, halogen, Ar¹, C1-C6 alkyl, C3-C6cycloalkyl, and C3-C6 heterocycloalkyl, wherein Ar¹ is either phenylsubstituted with 0-3 substituents independently selected from cyano,C1-C6 alkyl, C1-C6 haloalkyoxy, C1-C6 haloalkyl, and C1-C6polyhaloalkyl, C1-C6 cyanoalkyl, SO₂R¹², C1-C3 alkyl, C1-C3 alkylamine,and C1-C3 dialkylamino; or Ar¹ is monocyclic heteroaryl substituted with0-3 substituents independently selected from halo, cyano, C1-C6 alkyl,C1-C6 haloalkyoxy, C1-C6 haloalkyl, and C1-C6 polyhaloalkyl, C1-C6cyanoalkyl, SO₂R¹², C1-C3 alkyl, C1-C3 alkylamine, and C1-C3dialkylamino, wherein R¹² is selected from C1-C6 alkyl and C3-C6cycloalkyl; and

n is 0 or 1.

Substituents for compounds of structure (I′) are as defined in WO2012/135800.

In some embodiments, exemplary AXL kinase inhibitors are selected fromthe following, or a pharmaceutically acceptable salt thereof:

In certain more specific embodiments, the AXL kinase inhibitor is acompound of structure (I) or a pharmaceutically acceptable salt thereof.In certain embodiment, the AXL kinase inhibitor is the tartrate salt ofthe compound of structure (I) as disclosed herein.

In certain embodiments of the foregoing, the prior treatment regimencomprises treatment with a B-cell receptor signaling antagonist (e.g., aBruton's tyrosine kinase (BTK), SYK or PI3K inhibitor) or a Bcl-2inhibitor, or both. In other embodiments, the subject is ineligible fortreatment with a B-cell receptor signaling antagonist or a Bcl-2inhibitor, or both. In different embodiments, the subject is intolerantto treatment with a B-cell receptor signaling antagonist or a Bcl-2inhibitor, or both.

In certain embodiments, the B-cell receptor signaling antagonist is aBTK inhibitor. Exemplary BTK inhibitors, which may be included incertain embodiments of the prior treatment regimen, include those knownin the art, such as those described in PCT Pub. Nos: WO 2014/052365; WO2015/048689; WO 2015/002894; WO 2014/168975; WO 2014/159745; WO2014/130693; WO 2014/078578; WO 2014/018567; WO 2013/184572; WO2013/173518; WO 2013/116382; WO 2013/102059; WO 2013/059738; WO2013/010136; WO 2011/153514; WO 2011/046964; WO 2010/009342; WO2008/121742; WO 2008/054827; WO 2008/039218; WO 2007/087068; and in U.S.Pub. Nos: 2015/0018336; 2014/0336206; 2014/0243355; 2014/0212485;2014/0194446/2014/0187564; 2014/0135347; 2014/0128414; 2014/0187565;2014/0171453; 2014/0163027; 2014/01663046; 2014/0142126; 2014/0142123;2014/0128413; 2014/0079690; 2014/0080844; 2014/0057907; 2014/0039168;2013/0338172; 2013/0310402; 2013/0273030; 2013/0197014; 2013/0035334;2013/0012525; 2012/0283277; 2012/0283276; 2012/0277254; 2012/0252821;2010/0331350, the full disclosures of which are hereby incorporated byreference in their entireties.

In some embodiments, the BTK inhibitor is ONO-4059, AVL-292, SNS-062,CNX-774, CGI-1746, RN486 or ACP-196, which compounds are known in theart. In some specific embodiments, the BTK inhibitor is Ibrutinib.

In certain embodiments, the B-cell receptor signaling antagonist is aSYK inhibitor or a PI3Kgamma/delta inhibitor. In some embodiments, theSYK inhibitor is fostamatinib, entospletinib, cerdulatinib, or TAK-659.In some embodiments, the PI3Kgamma/delta inhibitor is idelalisib,duvelisib, TGR-1202, or ACP-319/AMG-319.

The Bcl-2 inhibitor, which may be included in certain embodiments of theprior treatment regimen, includes those known in the art. For example,in some of the foregoing embodiments, the BCL-2 inhibitor is Venetoclax(i.e., ABT-199). In some other embodiments the Bcl-2 inhibitor is anantisense oligonucleotide drug (e.g., oblimersen), a BH3 mimetic (e.g.,ABT-737, ABT-737-d8 or navitoclax/ABT-263 or ABT-263-d8), a novelnon-peptide inhibitor (e.g., TW-37), a pan-BCL-2 inhibitor (e.g.,Sabutoclax, Obatoclax), a Bcl-2 xl/BH3 domain interaction inhibitor(e.g., BH3I-1), a BCL-xl inhibitor (e.g., A-1331852 or A-1155463), anon-peptidic ligand of BCL-2 (e.g., HA14-1), a Bax activator (e.g.,BAM7), a small molecule BCL-2/BH4 domain antagonist (e.g., BDA-366), aflavonoid (e.g., Licochalcone A). In certain specific embodiments theBCL-2 inhibitor is FX1, AT-101, A-1210477, gambogic acid, UMI-77,Gossypol, (−)-Epigallocatechin Gallate, EM20-25, Nilotinib orNilotinib-d3, YC137, AG 1024, 3-bromopyruvic acid, Fluvastatin,Piperlongumine, 2,3-DCPE, 2-methoxy-antimycin A3 or Marinopyrrole A(i.e., Maritoclax).

In various different embodiments, the subject has greater than 0.01% MRDas determined by immunophenotypic (e.g., flow cytometry) or molecularbased (e.g., polymerase chain reaction and next-generation sequencing)analysis of a peripheral blood or bone marrow sample from the subject.In some embodiments, the subject has greater than 0.1% MRD as determinedby immunophenotypic or molecular based analysis of a peripheral blood orbone marrow sample from the subject. In different embodiments, thesubject has greater than 1.0% MRD as determined by immunophenotypic ormolecular based analysis of a peripheral blood or bone marrow samplefrom the subject. In still more embodiments, the subject has from 0.01%to 1.0% MRD as determined by immunophenotypic or molecular basedanalysis of a peripheral blood or bone marrow sample from the subject.

In some embodiments, MRD is determined based on % MRD in peripheralblood. In other embodiments, MRD is determined based on % MRD in bonemarrow.

The method for treating CLL with detectable MRD may further comprisedetermining the number of CLL cells and leukocytes in a peripheral bloodor bone marrow sample from the subject. The determining step can beperformed according to an in vitro method as known in the art anddescribed above. Alternatively, the determining is based on review ofdata obtained from an in vitro method described above.

The method may also further comprise categorizing the patient as havingMRD if the number of CLL cells per 10,000 leukocytes is greater than 1or categorizing the patient as having MRD if the number of CLL cells per10,000 leukocytes is from 1 to 100.

2. Biomarkers

In various embodiments, methods of the present disclosure comprisedetermining expression levels, tissue concentrations, or the presence ofone or more markers. In some embodiments, the markers comprise one ormore of Snail, Slug, AXL (e.g., AXL, Phospho AXL, Phospho/Total AXL,soluble AXL, etc.), AKT (e.g., Phospho/Total AKT), MMP, mTOR, PI3K, p38,JAK, STAT, SOCS1, SOCS3, PTEN, ERK, H2AX, CCNE, β-Catenin,P-Glycoprotein 1, GAS6, Retinoic acid, protein S, sMerTK, interleukin(IL)1β, IL2r/CD25, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL13, IL17,TNF-α, IFNγ, CSF2, CSF3, CCL2, sPD-L1/PD-L1, sPD-1/PD-1, sPD-L1/sPD-1,sCD163/CD163, E-cadherin, N-cadherin, Twist, FLT3, CDH1, CDH2, ZEB1,ADH1A, APOA2, ALDH1A2, CD38, CYP26A1, CYP26B1, DHRS3, DLX5, LGR1, FOXA1,HNP1B, HSD17B2, ISL1, LHX1, PPARG, RARA, RARB, RBP4, RXRG, SHH, STRA6,TGM2, and UCPI. In some embodiments, methods of the present disclosuremay comprise determining expression levels, tissue concentrations, orthe presence of one or more of ADH1A, APOA2, ALDH1A2, CD38, CYP26A1,CYP26B1, DHRS3, DLX5, LGR1, FOXA1, HNP1B, HSD17B2, ISL1, LHX1, PPARG,RARA, RARB, RBP4, RXRG, SHH, STRA6, TGM2, and UCPI. In particularembodiments, the one or more markers comprise AXL and/or GAS6. In someembodiments, the one or more markers comprise AXL. In particularembodiments, the one or more markers comprise soluble AXL. In someembodiments, the one or more markers comprise GAS6. In some embodiments,the one or more markers comprise soluble GAS6.

In some embodiments, methods of the present disclosure may comprisedetermining expression of markers of activated cells (e.g., dendriticcells, such as CD86, and CD11c). In some embodiments, the method of thepresent disclosure may comprise determining expression of markersspecific to detection of CLL including CD5, CD19, CD20, CD23, CD38,CD43, CD45, CD79b, CD81, kappa light chains, lambda light chains, andserum β2-microglobulin. In some embodiments, the method of the presentdisclosure may comprise determining mutations in genes associated withCLL including del(17p), TP53, and IGHV. In some embodiments, markerlevel is measured in any suitable tissue sample. For example, markerlevel may be measured in one or more of a subject's blood products(e.g., blood plasma, blood serum, peripheral blood mononuclear cells,tumor DNA species circulating as cell free DNA (cfDNA), circulatingtumor cells (CTC), circulating tumor DNA (ctDNA), circulating miRNA andthe like). In particular embodiments, the sample is a whole bloodsample, a serum sample, or a plasma sample. In specific embodiments, thesample is a whole blood sample. In other embodiments, the sample is aserum sample. In other embodiments, the sample is a plasma sample.

Expression levels, tissue concentrations, or the presence of markers maybe measured using any suitable techniques, such as enzyme-linkedimmunoassays (ELISA), mass spectrometry (MS), real-time quantitative PCR(RT-qPCR), flow cytometry, nucleic acid (i.e., DNA, RNA, etc.)sequencing, amino acid (i.e., peptide, protein, etc.) sequencing,molecular cytogenetics fluorescence in situ hybridization (FISH), andthe like.

In various embodiments, one or more of the markers described herein maybe up-regulated. “Up-regulation” or “up-regulated” refers to an increasein the presence of a protein and/or an increase in the expression of itsgene. In various embodiments, one or more markers may be down-regulated.“Down-regulation” or “down-regulated” refers to a decrease in thepresence of a protein and/or a decrease in the expression of its gene.Additionally, the function of a protein can be assayed by a relevantactivity assay. Exemplary activity assays include binding assays, enzymeactivity assays including, for example, protease assays, kinase assays,phosphatase assays, reductase assays, etc.

Up- or down-regulation of the markers can be assessed by comparing avalue to a relevant reference level. For example, the quantity of one ormore markers can be indicated as a value, which can be derived, e.g., bymeasuring level(s) of the marker(s) in the sample by an assay performed.In the broadest sense, the value may be qualitative or quantitative.Where detection is qualitative, the systems and methods provide areading or evaluation, e.g., assessment, of whether or not the marker ispresent in the sample being assayed. In yet other embodiments, thesystems and methods provide a quantitative detection of whether themarker is present in the sample being assayed, i.e., an evaluation orassessment of the actual amount or relative abundance of the marker inthe sample being assayed. In such embodiments, the quantitativedetection may be absolute or, if the method is a method of detecting twoor more different markers in a sample, relative. Accordingly, the term“quantifying” when used in the context of quantifying a marker in asample can refer to absolute or to relative quantification. Absolutequantification can be accomplished by inclusion of knownconcentration(s) of one or more control markers and referencing, e.g.,normalizing, the detected level of the marker with the known controlmarkers (e.g., through generation of a standard curve). Relativequantification can be accomplished by comparison of detected levels oramounts between the marker and a control (e.g., GAPDH or actin), toprovide a relative quantification of the marker, e.g., relative to thecontrol. Alternatively, relative quantification can be accomplished bycomparison of detected levels or amounts between two or more differentmarkers to provide a relative quantification of each of the two or moremarkers, e.g., relative to each other.

In some embodiments, the biomarker for identifying suitable patients tobe treated by any of the methods disclosed herein can be soluble AXL,GAS6, or a mensenchymal transcription factor. A patient having anelevated level of one or more of the biomarkers relative to a referencelevel may be identified as suitable for the treatment disclosed herein.A reference level can be the level of the corresponding biomarker in acontrol sample, which may be measured using the same method formeasuring the biomarker in a sample of a candidate patient. The controlmay be (or may be derived from) a normal subject (or normal subjects).Normal subjects, as used herein, refer to subjects that are apparentlyhealthy and show no signs or symptoms of a target cancer as disclosedherein. The population of control subjects may therefore be a populationof normal subjects. Alternatively, the control sample may be (or may bederived from) a subject or subjects having a target cancer who is/arenot responsive to one or more prior cancer therapies, e.g., thosedescribed herein. In some embodiments, the control sample may be (or maybe derived from) the subject being assessed for responsiveness to ananti-cancer therapy (e.g., those disclosed herein).

In specific examples, the biomarker is soluble AXL. The reference levelmay be a cutoff value of serum concentration of soluble AXL indicativeof disease prognosis and/or suitability to the treatment regimensdisclosed herein. In some examples, the cutoff value of soluble AXL canbe less than 41,000 pg/ml, which can be indicative of a high risk ofprogressive disease. In other examples, the cutoff value can be greaterthan about 42,000 pg/ml, which can be indicative of a likelihood ofstable disease. The cutoff values can be used to identify patientshaving specific prognostic features for treatment.

Measurement of the markers (i.e., the marker level) can be determined atthe protein or nucleic acid level using any method known in the art. Incertain embodiments, the measuring comprises measuring an mRNA level ora protein level. In particular embodiments, the measuring comprisesmeasuring an mRNA level.

In some embodiments, a marker is detected by contacting a sample withreagents (e.g., antibodies or nucleic acid primers), generatingcomplexes of reagent and marker(s), and detecting the complexes.Antibodies can be conjugated to a solid support suitable for adiagnostic assay in accordance with known techniques, such as passivebinding. Antibodies can be conjugated to cell surface antigens for adiagnostic assay in accordance with known techniques, such as flowcytometry, including multi-color flow cytometry. Antibodies or nucleicacid primers can be conjugated to detectable labels or groups such asradiolabels, enzyme labels, and fluorescent labels in accordance withknown techniques.

Examples of suitable immunoassays include immunoblotting,immunoprecipitation, immunofluorescence, chemiluminescence,electro-chemiluminescence (ECL), and ELISA. Up- or down-regulation ofmarkers also can be detected using, for example, cDNA arrays, clonehybridization, differential display, differential screening, FRETdetection, liquid microarrays, PCR, RT-PCR, Sanger sequencing,mass-parallel (next-generation) sequencing, molecular beacons,microelectric arrays, oligonucleotide arrays, polynucleotide arrays,serial analysis of gene expression (SAGE), and/or subtractivehybridization.

Examples of anti-AXL antibodies include AF154 (available from R&DSYSTEMS®), and MM0098-2N33 (available from ABCAM®). Examples ofanti-GAS6 antibodies include A-9 (available from SANTA CRUZBIOTECHNOLOGY®), and AF986 (available from R&D SYSTEMS®). Nucleic acidprimers may be designed using known techniques, based on the genesequence of AXL (NCBI Gene ID: 558) or GAS6 (NCBI Gene ID: 2621).

Marker level may be determined in a sample collected from a subjectprior to treatment. In such embodiments, marker levels may be used topredict responsiveness to a particular treatment. In some embodiments, asubject's marker level may be used to select an appropriate treatmentregimen. In some embodiments, levels may be used, at least in part, todetermine a treatment administered to a subject. In some embodiments, apre-treatment sample is collected on day 1 of a first cycle oftreatment, pre-dose. In some embodiments a pre-treatment sample may becollected one month, three weeks, two weeks, one week, six days, fivedays, four days, three days, two days, or one day prior toadministration of the first dose of the treatment regimen.

In embodiments, the sample is a whole blood sample, a serum sample, or aplasma sample. In certain embodiments, the expression level is an mRNAlevel or a protein level. In particular embodiments, the expressionlevel is an mRNA level.

In some embodiments, a sample may be collected after a dose of atreatment is administered to a subject. In specific embodiments, asample is collected on day 1 of a first cycle of treatment, predose, twohours after dosing, six hours after dosing, and 24 hours after dosing.In another specific embodiment, a sample is also collected on day 8 ofthe first cycle of treatment, predose. In another specific embodiment, asample is also collected on day 1 of the second cycle of treatment,predose. In further specific embodiment, a sample is also collected onday 1 of any additional cycles of treatment (e.g., third, fourth, fifth,etc.), predose. In another specific embodiment, a sample is alsocollected after treatment is completed.

A predetermined marker level may be measured from a sample collectedfrom a subject prior to treatment. In such embodiments, the marker levelmay be used to inform selection of a treatment regimen and/or predictresponsiveness to a particular treatment. In some embodiments, themarker level may be used, at least in part, to determine a treatmentregimen for a subject.

Accordingly, embodiments of the present disclosure include methods forselecting a treatment regimen for a subject in need thereof, the methodcomprising: obtaining a pre-treatment sample derived from the subject;measuring a marker in the sample; comparing the marker level to athreshold marker level; and selecting a treatment regimen based on thesame.

Embodiments of the present application include methods for selecting atreatment regimen for a subject in need thereof, the methods comprising:obtaining a pre-treatment sample derived from the subject; measuring amarker level in the sample; comparing the marker level to a thresholdmarker level; and selecting a treatment regimen comprising an effectiveamount of a compound of structure (I) or a pharmaceutically acceptablesalt thereof based on the comparison.

Further embodiments of the present application include methods forselecting a treatment regimen for a subject in need thereof, the methodscomprising: obtaining a pre-treatment sample derived from the subject;measuring a marker level of a marker in the sample; and selecting atreatment regimen comprising an effective amount of a compound ofstructure (I) or a pharmaceutically acceptable salt thereof if themarker level is above a threshold marker level.

Particular embodiments of the disclosure include methods for selecting atreatment regimen for a subject in need thereof, the method comprising:obtaining a pre-treatment sample derived from the subject; measuring amarker comprising AXL or GAS6 proteins in the sample; comparing themarker levels of the marker to a threshold marker level; and selecting atreatment regimen based on the comparison. In certain embodiments, thetreatment regimen comprises administering a pharmaceutical compositioncomprising a compound of structure (I) or a pharmaceutically acceptablesalt of a compound of structure (I) (e.g., a tartrate salt).

Further embodiments include administering a treatment regimen to asubject based on a predetermined level of a marker. Accordingly,embodiments of the disclosure include methods for treating a cancer in asubject, the method comprising: administering an effective amount of acompound of structure (I), or a pharmaceutically acceptable salt thereofto a subject having a predetermined level of a marker that is above athreshold marker level.

Further embodiments of the disclosure include methods for treating acancer in a subject, the method comprising: administering an effectiveamount of a compound of structure (I) or a pharmaceutically acceptablesalt thereof to a subject having a predetermined level of a markercomprising AXL or GAS6 proteins, wherein the predetermined level isabove a threshold marker level.

In some embodiments, the threshold marker level is associated with adisease outcome. Additionally, the threshold marker level may bedetermined from a population grouped according to disease outcome. Insome embodiments, the population was treated for cancer. In certainembodiments, the cancer was a solid tumor. In embodiments, the cancerwas a hematologic cancer. In particular embodiments, the population wastreated for the same type of disease (e.g., the same type of cancer) asthe subject.

In embodiments, the population has the same type of disease (e.g., thesame type of cancer) as the subject. In particular embodiments, thepopulation has the same type of cancer as the subject.

In some of the foregoing embodiments, the population had diseaseprogression following treatment with a compound of structure (I) or apharmaceutically acceptable salt thereof. Progressive disease may referto disease that has progressed in severity following initiation oftreatment. Accordingly, in particular embodiments, the threshold markerlevel is associated with a progressive disease outcome.

In some embodiments, the population had non-progression (e.g., stabledisease) following treatment with a compound of structure (I) orpharmaceutically acceptable salt thereof. Non-progression may refer todisease that has improved (e.g., remission) or disease that has notprogressed during the course of treatment (e.g., a tumor has notsignificantly grown or has decreased in size; and/or the cancer staginghas not increased or has decreased). In particular embodiments, thenon-progression is stable disease. Stable disease may refer to diseasethat has not improved but has not progressed during the course oftreatment. Accordingly, in other embodiments, the threshold marker levelis associated with non-progression.

In some embodiments, the population is a group comprising about 2, about5, about 10, about 25, about 50, about 75, or about 100 subjects. Insome embodiments, the population is a group comprising about 200, about300, about 500, about 1,000, about 1,500, about 2,000, about 3,000,about 5,000, or about 10,000 subjects. In some embodiments, thepopulation is a group comprising less than about 10,000 subjects. Inother embodiments, the population is a group comprising greater thanabout 10,000 subjects.

In certain embodiments, the subject's marker level is higher than athreshold marker level associated with progressive disease. Inembodiments, the subject's marker level is greater than or equal to athreshold marker level associated with non-progression.

In embodiments, the threshold marker level of AXL protein is about35,000 picograms per milliliter (pg/mL). In some embodiments, thethreshold marker level of AXL protein is about 36,000 pg/mL. In someembodiments, the threshold marker level of AXL protein is about 37,000pg/mL. In some embodiments, the threshold marker level of AXL protein isabout 37,500 pg/mL. In some embodiments, the threshold marker level ofAXL protein is about 38,000 pg/mL. In some embodiments, the thresholdmarker level of AXL protein is about 38,500 pg/mL. In some embodiments,the threshold marker level of AXL protein is about 39,000 pg/mL. In someembodiments, the threshold marker level of AXL protein is about 39,500pg/mL. In some embodiments, the threshold marker level of AXL protein isabout 40,000 pg/mL. In some embodiments, the threshold marker level ofAXL protein is about 40,500 pg/mL. In some embodiments, the thresholdmarker level of AXL protein is about 41,000 pg/mL. In some embodiments,the threshold marker level of AXL protein is about 41,500 pg/mL. In someembodiments, the threshold marker level of AXL protein is about 42,000pg/mL. In particular embodiments, the threshold marker level of AXLprotein is about 43,000 pg/mL. In some embodiments, the threshold markerlevel of AXL protein is about 44,000 pg/mL. In certain embodiments, thethreshold marker level of AXL protein is about 45,000 pg/mL. In someembodiments, the threshold marker level of AXL protein is about 46,000pg/mL. In some embodiments, the threshold marker level of AXL protein isabout 47,000 pg/mL.

In some embodiments, the threshold marker level of GAS6 protein is about8,000 pg/mL. In some embodiments, the threshold marker level of GAS6protein is about 9,000 pg/mL. In some embodiments, the threshold markerlevel of GAS6 protein is about 10,000 pg/mL. In particular embodiments,the threshold marker level of GAS6 protein is about 10,500 pg/mL. Insome embodiments, the threshold marker level of GAS6 protein is about11,000 pg/mL. In particular embodiments, the threshold marker level ofGAS6 protein is about 11,500 pg/mL. In some embodiments, the thresholdmarker level of GAS6 protein is about 12,000 pg/mL. In certainembodiments, the threshold marker level of GAS6 protein is about 12,500pg/mL. In particular embodiments, the threshold marker level of GAS6protein is about 13,000 pg/mL. In some embodiments, the threshold markerlevel of GAS6 protein is about 13,500 pg/mL. In some embodiments, thethreshold marker level of GAS6 protein is about 14,000 pg/mL. In certainembodiments, the threshold marker level of GAS6 protein is about 14,500pg/mL. In particular embodiments, the threshold marker level of GAS6protein is about 15,000 pg/mL. In particular embodiments, the thresholdmarker level of GAS6 protein is about 15,500 pg/mL. In particularembodiments, the threshold marker level of GAS6 protein is about 16,000pg/mL. In certain embodiments, the threshold marker level of GAS6protein is about 16,500 pg/mL.

Accordingly, embodiments of the present disclosure include methods ofselecting a treatment regimen for a cancer in a subject, the methodcomprising: obtaining a pre-treatment sample derived from the subject;measuring the level of a marker comprising AXL or GAS6 in the sample;and selecting a treatment regimen comprising an effective amount of anAXL inhibitor if: (A) the level of AXL protein is at least about 35,000picograms per milliliter (pg/mL); (B) the level of GAS6 protein is atleast about 8,000 pg/mL; or (C) both.

Accordingly, embodiments of the present disclosure include methods ofselecting a treatment regimen for a cancer in a subject, the methodcomprising: obtaining a pre-treatment sample derived from the subject;measuring the level of a marker comprising AXL or GAS6 in the sample;and selecting a treatment regimen comprising an effective amount of anAXL inhibitor if: (A) the level of AXL protein is at least about 45,000picograms per milliliter (pg/mL); (B) the level of GAS6 protein is atleast about 13,500 pg/mL; or (C) both.

Particular embodiments of the present disclosure include methods ofselecting a treatment regimen for a cancer in a subject, the methodcomprising: obtaining a pre-treatment sample derived from the subject;measuring the level of a marker comprising AXL or GAS6 in the sample;and selecting a treatment regimen comprising an effective amount of acompound of structure (I):

or a pharmaceutically acceptable salt thereof if: (A) AXL protein ispresent at a level of at least about 45,000 pg/mL; (B) GAS6 protein ispresent at a level of at least about 13,500 pg/mL; or (C) both

In embodiments, a subject's marker level is pre-determined.

Accordingly, embodiments of the present disclosure include methods fortreating a cancer in a subject, the method comprising: administering aneffective amount of an AXL inhibitor to a subject having a predeterminedlevel of a marker comprising AXL or GAS6, wherein: (A) the predeterminedlevel of AXL protein is at least 45,000 pg/mL; (B) the predeterminedlevel of GAS6 protein is at least 13,500 pg/mL; or (C) both.

Further embodiments of the present disclosure include methods fortreating a cancer in a subject, the method comprising: administering aneffective amount of a compound of structure (1):

or a pharmaceutically acceptable salt thereof to a subject having apredetermined level of a marker comprising AXL or GAS6, wherein: (A) thepredetermined level of AXL protein is at least 35,000 pg/mL; (B) thepredetermined level of GAS6 protein is at least 8,000 pg/mL; or (C)both.

In still further embodiments of the present disclosure include methodsfor treating a cancer in a subject, the method comprising: administeringan effective amount of a compound of structure (I):

or a pharmaceutically acceptable salt thereof to a subject having apredetermined level of a marker comprising AXL or GAS6, wherein: (A) thepredetermined level of AXL protein is at least 45,000 pg/mL; (B) thepredetermined level of GAS6 protein is at least 13,500 pg/mL; or (C)both.

3. Combination Therapies

In still another embodiment, an AXL kinase inhibitor, such as a compoundof structure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt), is administered to a subject inneed thereof in combination with a B-cell receptor signaling antagonist(e.g., a Bruton's tyrosine kinase (BTK) inhibitor, such as Ibrutinib).Accordingly, methods of the present disclosure include methods fortreating cancer comprising administering an effective amount of an AXLkinase inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor to asubject in need thereof. The administration may be before, concurrentlyor after administration of the B-cell receptor signaling antagonist(e.g., the BTK inhibitor).

In some embodiments, the AXL kinase inhibitor and BTK inhibitor areco-administered. In other embodiments, the AXL kinase inhibitor isadministered after the BTK inhibitor. In still different embodiments,the AXL kinase inhibitor is administered before the BTK inhibitor.

In various embodiments, the BTK inhibitor is Ibrutinib. In someparticular embodiments, the cancer is chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), or both. In some embodiments,the subject has received a prior treatment regimen for CLL, SLL, orboth. In some embodiments, the subject was refractory after the priortreatment regimen, the subject has relapsed CLL, SLL, or both after aresponse to the prior treatment regimen, or the subject has detectableminimal residual disease (MRD).

In certain embodiments the subject is insensitive to treatment with aB-cell receptor signaling antagonist (e.g., a BTK inhibitor), isineligible for treatment with a B-cell receptor signaling antagonist(e.g., a BTK inhibitor) or has relapsed after treatment with a B-cellreceptor signaling antagonist (e.g., a BTK inhibitor). In one specificembodiment, a compound of structure (I) or a pharmaceutically acceptablesalt of a compound of structure (I) (e.g., a tartrate salt), isadministered to a subject in need thereof in combination with a BTKinhibitor, such as Ibrutinib for treatment of leukemia (e.g., CLL, SLL,or both).

In another embodiment, an AXL kinase inhibitor, such as a compound ofstructure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt), is administered to a subject inneed thereof in combination with a Bcl-2 inhibitor, such as venetoclax.The administration may be before, concurrently or after administrationof the Bcl-2 inhibitor. In certain embodiments the subject isinsensitive to treatment with a Bcl-2 inhibitor, is ineligible fortreatment with a Bcl-2 inhibitor or has relapsed after treatment with aBcl-2 inhibitor In one specific embodiment, a compound of structure (I)or a pharmaceutically acceptable salt of a compound of structure (I)(e.g., a tartrate salt), is administered to a subject in need thereof incombination with a Bcl-2 inhibitor, such as venetoclax for treatment ofleukemia (e.g., CLL, SLL, or both).

In still another embodiment, an AXL kinase inhibitor, such as a compoundof structure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt), is administered to a subject inneed thereof in combination with an immune checkpoint inhibitor (e.g., aPD-1 inhibitor (such as Pembrolizumab or Nivolumab), a PD-L1 inhibitor(such as Atezolizumab, Avelumab, or Durvalumab), a CTLA-4 inhibitor, aLAG-3 inhibitor, or a Tim-3 inhibitor). Accordingly, methods of thepresent disclosure include methods for treating cancer comprisingadministering an effective amount of an AXL kinase inhibitor and animmune checkpoint inhibitor to a subject in need thereof. Theadministration of the AXL kinase inhibitor may be before, concurrentlyor after administration of the immune checkpoint inhibitor (e.g., a PD-1inhibitor (such as Pembrolizumab or Nivolumab), a PD-L1 inhibitor (suchas Atezolizumab, Avelumab, or Durvalumab), a CTLA-4 inhibitor, a LAG-3inhibitor, or a Tim-3 inhibitor).

In some embodiments, the AXL kinase inhibitor and immune checkpointinhibitor are co-administered. In other embodiments, the AXL kinaseinhibitor is administered after the immune checkpoint inhibitor. Instill different embodiments, the AXL kinase inhibitor is administeredbefore the immune checkpoint inhibitor.

In various embodiments, the immune checkpoint inhibitor is a PD-1inhibitor. In specific embodiments, the PD-1 inhibitor is Pembrolizumab,Nivolumab, or a combination thereof. In particular embodiments, the PD-1inhibitor is Pembrolizumab. In particular embodiments, the PD-1inhibitor is Nivolumab. In some other embodiments, the PD-1 inhibitor isCBT-501 (CBT Pharmaceuticals), CBT-502 (CBT Pharmaceuticals), JS001(Junshi Biosciences), IBI308 (Innovent Biologies), SHR-1210 (HengruiMedicine), BGB-A317 (Beigene), BAT-1306 (Bio-Thera Solutions), GLS-010(Gloria Pharmaceuticals; WuXi Biologies), AK103, AK104, AK105 (AkesioBiopharma; Hangzhou Hansi Biologies; Hanzhong Biologies), LZM009(Livzon), HLX-10 (Henlius Biotech), or CS1003 (CStone Pharmaceuticals).In some embodiments, the PD-1 inhibitor is a monoclonal antibody (e.g.,made by Genor Biopharma and in Phase I of clinical trials as of thisfiling; as made by Shenzhou Gongcheng and applying for clinical trialsas of this filing; as made by Lunan Hope Pharmaceuticals and applyingfor clinical trials as of this filing).

In some embodiments, the immune checkpoint inhibitor is a PD-L1inhibitor. In some such embodiments, the PD-L1 inhibitor isAtezolizumab, Avelumab, Durvalumab, or a combination thereof. Inparticular embodiments, the PD-L1 inhibitor is Atezolizumab. Inparticular embodiments, the PD-L1 inhibitor is Avelumab. In particularembodiments, the PD-L1 inhibitor is Durvalumab. In certain embodiments,the PD-L1 inhibitor is KN035 (Alphamab; 3DMed), CS1001 (CStonePharmaceuticals), SHR-1316 (Hengrui Medicine), TQB2450 (ChiataiTianqing), STI-A1014 (Zhaoke Pharm; Lee's Pharm), BGB-A333 (Beigene),MSB2311 (Mabspace Biosciences), or HLX-20 (Henlius Biotech). In someembodiments, the PD-L1 inhibitor is a monoclonal antibody (e.g., as madeby Hisun Pharm and applying for clinical trials as of this filing).

In some embodiments, the immune checkpoint inhibitor is a CTLA-4inhibitor. In certain embodiments, the CTLA-4 inhibitor is ipilimumab.In other embodiments, the CTLA-4 inhibitor is tremelimumab.

In embodiments, an AXL kinase inhibitor, such as a compound of structure(I) or a pharmaceutically acceptable salt of a compound of structure (I)(e.g., a tartrate salt), is administered to a subject in need thereof incombination with a bromodomain inhibitor, a histone deacetylase (HDAC),or both.

A bromodomain inhibitor inhibits at least one bromodomain protein, suchas Brd2, Brd3, Brd4 and/or BrdT, for example Brd4. In some of theseembodiments, the bromodomain inhibitor is JQ-1 (Nature 2010 Dec. 23;468(7327): 1067-73), BI2536 (ACS Chem. Biol. 2014 May 16; 9(5): 1160-71;Boehringer Ingelheim), TG101209 (ACS Chem. Biol. 2014 May 16; 9(5):1160-71), OTX015 (Mol. Cancer Ther. November 201312; C244; Oncoethix),IBET762 (J Med Chem. 2013 Oct. 10; 56(19):7498-500; GlaxoSmithKline),IBET151 (Bioorg. Med. Chem. Lett. 2012 Apr. 15; 22(8):2968-72;GlaxoSmithKline), PFI-1 (J. Med. Chem. 2012 Nov. 26; 55(22):9831-7;Cancer Res. 2013 Jun. 1; 73(11):3336-46; Structural Genomics Consortium)of CPI-0610 (Constellation Pharmaceuticals). In some embodiments, thebromodomain inhibitor is TG101209, BI2536, OTX015, C244, IBET762,IBET151, or PFI-1.

A HDAC inhibitor inhibits at least one HDAC protein. HDAC proteins maybe grouped into classes based on homology to yeast HDAC proteins withClass I made up of HDAC1, HDAC2, HDAC3 and HDAC 8; Class Ha made up ofHDAC4, HDAC5, HDAC7 and HDAC 9; Class lib made up of HDAC 6 and HDAC10;and Class IV made up of HDAC11. In some of these embodiments, the HDACinhibitor is trichostatin A, vorinostat (Proc. Natl. Acad. Sci. U.S.A.1998 Mar. 17; 95(6):3003-7), givinostat, abexinostat (Mol. Cancer Ther.2006 May; 5(5):1309-17), belinostat (Mol. Cancer Ther. 2003 August;2(8):721-8), panobinostat (Clin. Cancer Res. 2006 Aug. 1;12(15):4628-35), resminostat (Clin. Cancer Res. 2013 Oct. 1;19(19):5494-504), quisinostat (Clin. Cancer Res. 2013 Aug. 1;19(15):4262-72), depsipeptide (Blood. 2001 Nov. 1; 98(9):2865-8),entinostat (Proc. Natl. Acad. Sci. U.S.A. 1999 Apr. 13; 96(8):4592-7),mocetinostat (Bioorg. Med. Chem. Lett. 2008 Feb. 1; 18(3): 1067-71) orvalproic acid (EMBO J. 2001 Dec. 17; 20(24):6969-78). For example, insome embodiments the HDAC inhibitor is panobinostat, vorinostat, MS275,belinostat, or LBH589. In some embodiments, the HDAC inhibitor ispanobinostat or SAHA.

In some embodiments, methods of the present disclosure further compriseadministering radiation therapy to the subject.

In some of the foregoing embodiments, the method is for treating livercancer, refractory cancers (e.g., non-small cell lung cancer), lungcancer, esophageal cancer, Hodgkin's lymphoma, NK/T-cell lymphoma, ormelanoma. In some specific embodiments, the method is for treatingesophageal squamous cell carcinoma, gastric cancer, lung cancer,nasopharyngeal carcinoma, bladder cancer, soft tissue sarcoma, diffuselarge B-cell lymphoma, head and neck squamous cell carcinomas, kidneycancer, urothelial carcinoma, ovarian cancer, uterine cancer, orpancreatic cancer.

Other embodiments provide methods for combination therapies in which anagent known to modulate other pathways, or other components of the samepathway, or even overlapping sets of target enzymes are used incombination with a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt).In one aspect, such therapy includes but is not limited to thecombination of a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)with chemotherapeutic agents, therapeutic antibodies, and radiationtreatment, to provide a synergistic or additive therapeutic effect.

Many chemotherapeutics are presently known in the art and can be used incombination with a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt).In some embodiments, the chemotherapeutic is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomeRASe inhibitors, biological responsemodifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.

Non-limiting examples of therapeutic agents that can be used incombinations with a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)are chemotherapeutic agents, cytotoxic agents, and non-peptide smallmolecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib),Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin as well as ahost of chemotherapeutic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, Casodex®, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.,paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); retinoicacid; esperamicins; capecitabine; and pharmaceutically acceptable salts,acids or derivatives of any of the above.

Also included as suitable chemotherapeutic cell conditioners areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,(Nolvadex™), raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda;ibandronate; camptothecin-11 (CPT-11); topoisomeRASe inhibitor RFS 2000;difluoromethylomithine (DMFO). Where desired, the compound of structure(I) or a pharmaceutically acceptable salt of a compound of structure (I)(e.g., a tartrate salt) or a pharmaceutical composition thereof can beused in combination with commonly prescribed anti-cancer drugs such asHerceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®,ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab,17-N-Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib,3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide,Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenicherbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine,BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine,CBV (chemotherapy), Calyculin, cell-cycle nonspecific antineoplasticagents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine,Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol,Forodesine, Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon,Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel,Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide,Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone,Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38,Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin,Tariquidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin tetranitrate,Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan,Vinflunine, ZD6126 or Zosuquidar.

In one embodiment, a compound of structure (I), is administered to asubject in need thereof in combination with a CDK9 inhibitor, such asAlvocidib. In a related embodiment, a pharmaceutically acceptable saltof a compound of structure (I) (e.g., a tartrate salt) is administeredto a subject in need thereof in combination with a CDK9 inhibitor, suchas Alvocidib. The administration may be before, concurrently or afteradministration of the CDK9 inhibitor. In one specific embodiment, acompound of structure (I) is administered to a subject in need thereofin combination with a CDK9 inhibitor, such as Alvocidib for treatment ofpancreatic cancer. In a related specific embodiment, a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)is administered to a subject in need thereof in combination with a CDK9inhibitor, such as Alvocidib for treatment of pancreatic cancer. In someof the foregoing embodiments, the salt is a tartrate salt. In some ofthe foregoing embodiments, the CDK9 inhibitor is Alvocidib. In someembodiments, the salt is a tartrate salt and the CDK9 inhibitor isAlvocidib.

In certain other embodiments, a method for treating cancer is provided,the method comprising administering an effective amount of an AXL kinaseinhibitor and a CDK inhibitor to a subject in need thereof. The AXLkinase inhibitor and CDK inhibitor may be any of the AXL kinase or CDKinhibitors known in the art.

In certain embodiments, the AXL kinase inhibitor is a compound ofstructure (I), or a pharmaceutically acceptable salt thereof. Forexample, in some specific embodiments, the AXL kinase inhibitor atartrate salt of a compound of structure (I) as disclosed herein.

In embodiments, the CDK inhibitor is a CDK2, CDK4, CDK6, CDK7, CDK8,CDK9, CDK10, and/or CDK11 inhibitor. In some embodiments, the CDKinhibitor is a CDK7, CDK9 inhibitor, or both. In some embodiments, theCDK inhibitor is dinaciclib (ACS Med. Chem. Lett. 2010 May 17;1(5):204-8; Mol. Cancer Ther. 2010 August; 9(8):2344-53; Merck, Sharpand Dohme), AT7519 (J. Med. Chem. 2008 Aug. 28; 51(16):4986-99; AstexPharmaceutical) or palbociclib (J. Med. Chem. 2005 Apr. 7;48(7):2388-406; Pfizer). In certain embodiments, the CDK inhibitor is aCDK9 inhibitor, such as alvocidib. The alvocidib may be administered asthe free bases, as a pharmaceutically acceptable salt or as a prodrug.In certain embodiments, the CDK9 inhibitor is alvocidib. in otherembodiments, the CDK9 inhibitor is a pharmaceutically acceptable salt ofalvocidib. In other embodiments, the CDK9 inhibitor is a prodrug ofalvocidib. Prodrugs of alvocidib include those disclosed in WO2016/187316, the full disclosure of which is hereby incorporated byreference in its entirety. For example, in some embodiments the prodrugof alvocidib has the following structure (II):

or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof,wherein: one of R¹, R² or R³ is —P(═O)(OH)₂, and the other two of R¹, R²and R³ are each H.

In certain specific embodiments, the prodrug of alvocidib has thefollowing structure:

or a pharmaceutically acceptable salt or zwitterionic form thereof.

In some embodiments, the AXL kinase inhibitor and CDK inhibitor areco-administered. In other embodiments, the AXL kinase inhibitor isadministered after the CDK inhibitor. In still different embodiments,the AXL kinase inhibitor is administered before the CDK inhibitor.

Various different cancers can be treated with the combination of an AXLkinase inhibitor and CDK inhibitor. In some embodiments, the cancer is ahematologic cancer or solid tumor, for example any of the hematologiccancers or solid tumors disclosed herein or known in the art.

In some specific embodiments, the cancer is a hematologic cancer, suchas multiple myeloma, myelodysplastic syndrome (MDS), acute myelogenousleukemia (AML), acute lymphoblastic leukemia (ALL), acute lymphocyticleukemia, chronic lymphogenous leukemia, chronic lymphocytic leukemia(CLL), mantle cell lymphoma, diffuse large B-cell lymphoma, follicularlymphoma, or non-Hodgkin's lymphoma. In some specific embodiments, thehematologic cancer is CLL, SLL, or both. In some specific embodiments,the hematologic cancer is CLL. In some specific embodiments, thehematologic cancer is SLL.

In some other specific embodiments, the cancer treated by thecombination of an AXL kinase inhibitor and a CDK inhibitor is a solidtumor, such as a pancreatic, colon or lung cancer.

Embodiments further relate to a method of administering a compound ofstructure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt), to a subject in need thereof incombination with a BTK inhibitor (e.g., Ibrutinib) or a CDK9 inhibitor(e.g., Alvocidib) provided herein, in combination with radiation therapyfor inhibiting abnormal cell growth or treating the hyperproliferativedisorder in the mammal. Techniques for administering radiation therapyare known in the art, and these techniques can be used in thecombination therapy described herein. The administration of apharmaceutically acceptable salt of a compound of structure (I), forexample a tartrate salt of a compound of structure (I) in thiscombination therapy can be determined as described herein.

In one embodiment, a compound of structure (I), is administered to asubject in need thereof in combination with an ATR inhibitor, such asAZD6738 or VX-970. In a related embodiment, a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)is administered to a subject in need thereof in combination with an ATRinhibitor, such as AZD6738 or VX-970. The administration may be before,concurrently or after administration of the ATR inhibitor. In onespecific embodiment, a compound of structure (I) is administered to asubject in need thereof in combination with an ATR inhibitor, such asAZD6738 or VX-970 for treatment of non-small cell lung cancer. In arelated specific embodiment, a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt) is administered to asubject in need thereof in combination with an ATR inhibitor, such asAZD6738 or VX-970 for treatment of non-small cell lung cancer. In someof the foregoing embodiments, the salt is a tartrate salt. In some ofthe foregoing embodiments, the ATR inhibitor is AZD6738. In some of theforegoing embodiments, the ATR inhibitor is VX-970. In some embodiments,the salt is a tartrate salt and the ATR inhibitor is AZD6738. In someembodiments, the salt is a tartrate salt and the ATR inhibitor isVX-970. In some of the foregoing embodiments, the ATR inhibitor is acombination of AZD6738 and VX-970.

In some of the foregoing embodiments, the non-small cell lung cancercomprises TCGA lung adenocarcinoma, one or more LUAD tumors, TCGA lungsquamous cell carcinoma, one or more LUSC tumors, one or more MDACCPROSPECT tumors, one or more MDACC BATTLE1 tumors, one or more BATTLE2tumors, or combinations thereof. In some embodiments, the non-small celllung cancer comprises TCGA LUAD tumors, for example, tumors enriched inALK translocations. In some embodiments, the non-small cell lung cancercomprises TCGA LUAD tumors, for example, tumors comprising one or moreEGER mutations.

In one embodiment, a compound of structure (I), is administered to asubject in need thereof thereby sensitizing the subject toadministration of an ATR inhibitor, such as AZD6738 or VX-970. In arelated embodiment, a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt) is administered to a subject inneed thereof thereby sensitizing the subject to administration of an ATRinhibitor, such as AZD6738 or VX-970. In one specific embodiment, acompound of structure (I) is administered to a subject in need thereofthereby sensitizing the subject to administration of an ATR inhibitor,such as AZD6738 or VX-970 for treatment of non-small cell lung cancer.In a related specific embodiment, a pharmaceutically acceptable salt ofa compound of structure (I) (e.g., a tartrate salt) is administered to asubject in need thereof thereby sensitizing the subject toadministration of an ATR inhibitor, such as AZD6738 or VX-970 fortreatment of non-small cell lung cancer. In some of the foregoingembodiments, the salt is a tartrate salt. In some of the foregoingembodiments, the ATR inhibitor is AZD6738. In some of the foregoingembodiments, the ATR inhibitor is VX-970. In some embodiments, the saltis a tartrate salt and the ATR inhibitor is AZD6738. In someembodiments, the salt is a tartrate salt and the ATR inhibitor isVX-970. In some of the foregoing embodiments, the ATR inhibitor is acombination of AZD6738 and VX-970.

Radiation therapy can be administered in combination with a compound ofstructure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt) in some embodiments. Exemplaryradiation therapies include external-beam therapy, internal radiationtherapy, implant radiation, stereotactic radiosurgery, systemicradiation therapy, radiotherapy and permanent or temporary interstitialbrachytherapy. The term “brachytherapy,” as used herein, refers toradiation therapy delivered by a spatially confined radioactive materialinserted into the body at or near a tumor or other proliferative tissuedisease site. The term is intended without limitation to includeexposure to radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶,Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactive isotopes of Lu). Suitableradiation sources for use as a cell conditioner of the present inventioninclude both solids and liquids. By way of non-limiting example, theradiation source can be a radionuclide, such as I¹²⁵, I¹³¹, Yb¹⁶⁹, Ir¹⁹²as a solid source, I¹²⁵ as a solid source, or other radionuclides thatemit photons, beta particles, gamma radiation, or other therapeuticrays. The radioactive material can also be a fluid made from anysolution of radionuclide(s), e.g., a solution of I¹²⁵ or I¹³¹, or aradioactive fluid can be produced using a slurry of a suitable fluidcontaining small particles of solid radionuclides, such as Au¹⁹⁸, Y⁹⁰.Moreover, the radionuclide(s) can be embodied in a gel or radioactivemicro spheres.

Without being limited by any theory, a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt) can render abnormal cells more sensitive to treatmentwith radiation for purposes of killing and/or inhibiting the growth ofsuch cells. Accordingly, some embodiments include a method forsensitizing abnormal cells in a mammal to treatment with radiation whichcomprises administering to the mammal an amount of a compound ofstructure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt), which amount is effective issensitizing abnormal cells to treatment with radiation. The amount of acompound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt) in this method can bedetermined according to the means for ascertaining effective amounts ofsuch compounds and salts described herein.

The compound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt) can also be used incombination with an amount of one or more substances selected fromanti-angiogenesis agents, signal transduction inhibitors,antiproliferative agents, glycolysis inhibitors, or autophagyinhibitors.

Anti-angiogenesis agents include, for example, MMP-2(matrix-metalloproteinase 2) inhibitors, rapamycin, temsirolimus(CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab.Examples of useful COX-II inhibitors include CELEBREX™ (alecoxib),valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinaseinhibitors are described in WO 96/33172 (published Oct. 24, 1996), WO96/27583 (published Mar. 7, 1996), European Patent Application No.97304971.1 (filed Jul. 8, 1997), European Patent Application No.99308617.2 (filed Oct. 29, 1999), WO 98/07697 (published Feb. 26, 1998),WO 98/03516 (published Jan. 29, 1998), WO 98/34918 (published Aug. 13,1998), WO 98/34915 (published Aug. 13, 1998), WO 98/33768 (publishedAug. 6, 1998), WO 98/30566 (published Jul. 16, 1998), European PatentPublication 606,046 (published Jul. 13, 1994), European PatentPublication 931, 788 (published Jul. 28, 1999), WO 90/05719 (publishedMay 31, 1990), WO 99/52910 (published Oct. 21, 1999), WO 99/52889(published Oct. 21, 1999), WO 99/29667 (published Jun. 17, 1999), PCTInternational Application No. PCT/IB98/01113 (filed Jul. 21, 1998),European Patent Application No. 99302232.1 (filed Mar. 25, 1999), GreatBritain Patent Application No. 9912961.1 (filed Jun. 3, 1999), U.S.Provisional Application No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat.No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issuedJan. 19, 1999), and European Patent Publication 780,386 (published Jun.25, 1997), all of which are incorporated herein in their entireties byreference. Embodiments of MMP-2 and MMP-9 inhibitors include those thathave little or no activity inhibiting MMP-1. Other embodiments includethose that selectively inhibit MMP-2 and/or AMP-9 relative to the othermatrix-metalloproteinases (i.e., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specificexamples of MMP inhibitors useful in some embodiments are AG-3340, RO32-3555, and RS 13-0830.

Autophagy inhibitors include, but are not limited to chloroquine,3-methyladenine, hydroxychloroquine (Plaquenil™), bafilomycin A1,5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid,autophagy-suppressive algal toxins which inhibit protein phosphatases oftype 2A or type 1, analogues of cAMP, and drugs which elevate cAMPlevels such as adenosine, LY204002, N6-mercaptopurine riboside, andvinblastine. In addition, antisense or siRNA that inhibits expression ofproteins including but not limited to ATG5 (which are implicated inautophagy), may also be used.

In other embodiments, agents useful in methods for combination therapywith a compound of structure (I) or a pharmaceutically acceptable saltof a compound of structure (I) (e.g., a tartrate salt) include, but arenot limited to: Erlotinib, Afatinib, Iressa, GDC0941, MLN1117, BYL719(Alpelisib), BKM120 (Buparlisib), CYT387, GLPG0634, Baricitinib,Lestaurtinib, momelotinib, Pacritinib, Ruxolitinib, TG101348,Crizotinib, tivantinib, AMG337, cabozantinib, foretinib, onartuzumab,NVP-AEW541, Dasatinib, Ponatinib, saracatinib, bosutinib, trametinib,selumetinib, cobimetinib, PD0325901, RO5126766, Axitinib, Bevacizumab,Bostutinib, Cetuximab, Crizotinib, Fostamatinib, Gefitinib, Imatinib,Lapatinib, Lenvatinib, Ibrutinib, Nilotinib, Panitumumab, Pazopanib,Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib, Sunitinib, SU6656,Trastuzumab, Tofacitinib, Vandetanib, Vemurafenib, Irinotecan, Taxol,Docetaxel, Rapamycin or MLN0128.

In embodiments, a compound of structure (I) or a pharmaceuticallyacceptable salt of a compound of structure (I) (e.g., a tartrate salt)is administered in combination with an epidermal growth factor receptortyrosine kinase (EGFR) inhibitor. Examples of EGFR inhibitors includeerlotinib, osimertinib, cetuximab, gefitinib, necitumumab, lapatinib,neratinib, panitumumab, vandetanib, and necitumumab. A combination of acompound of structure (I) and an EGFR inhibitor may be useful, forexample, in the treatment of cancers that are related to EGFRdysregulation, such as non-small-cell lung cancer (NSCLC), pancreaticcancer, breast cancer, and colon cancer. EGFR may be dysregulated, forexample, due to activating mutations in exons 18, 19, 20, or 21. Asshown in Examples 9, 10, and 19, combinations of a compound of structure(I) and EGFR inhibitors show synergy in preclinical models ofEGFR-mutated cancer. In particular embodiments, the EGFR inhibitor iserlotinib or osimertinib. In particular embodiments, the combination ofa compound of structure (I) and an EGFR inhibitor is used to treatEGFR-mutated NSCLC. In particular embodiments, the combination of acompound of structure (I) and an EGFR inhibitor is used to treat an EGFRinhibitor-resistant cancer, and the compound of structure (I) sensitizedthe cancer to the EGFR inhibitor.

In certain embodiments, a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt) is administered in combination with Erlotinib. In someembodiments, such a combination is used to treat pancreatic cancer. Inother embodiments, such a combination is used to treat lung cancer. Infurther embodiments, the lung cancer is non-small cell lung cancer.

In certain embodiments, a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt) is administered in combination with osmertinib. In someembodiments, such a combination is used to treat lung cancer. In furtherembodiments, the lung cancer has an EGFR mutation.

When used in combination therapy, a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt), is administered with the second agent simultaneously orseparately. This administration in combination can include simultaneousadministration of the two agents in the same dosage form, simultaneousadministration in separate dosage forms, and separate administration.That is, a compound of structure (I) or a pharmaceutically acceptablesalt of a compound of structure (I) (e.g., a tartrate salt), and any ofthe agents described above (e.g., Ibrutinib or Alvocidib) can beformulated together in the same dosage form and administeredsimultaneously. Alternatively, a compound of structure (I) or apharmaceutically acceptable salt of a compound of structure (I) (e.g., atartrate salt), and any of the agents described above (e.g., Ibrutinibor Alvocidib) can be simultaneously administered, wherein both theagents are present in separate formulations. In another alternative, acompound of structure (I) or a pharmaceutically acceptable salt of acompound of structure (I) (e.g., a tartrate salt), can be administeredjust followed by and any of the agents described above, or vice versa.In some embodiments of the separate administration protocol, a compoundof structure (I) or a pharmaceutically acceptable salt of a compound ofstructure (I) (e.g., a tartrate salt), and any of the agents describedabove are administered a few minutes apart, or a few hours apart, or afew days apart.

A compound of structure (I) or a pharmaceutically acceptable saltthereof (e.g., a tartrate salt) used in embodiments of the disclosuremay also be administered simultaneously with, prior to, or afteradministration of one or more other therapeutic agents. For example, acompound of structure (I) or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt) can be administered and after a sufficientperiod of time a second therapeutic agent is administered. In suchembodiments, the period of time between the administration of a compoundof structure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) and the second therapeutic agent may be referred to as a“treatment break.” In some embodiments, such a treatment break rangesfrom about 12 hours to about 48 hours. In some embodiments, such atreatment break ranges from about 18 to about 40 hours. In someembodiments, such a treatment break ranges from about 18 to about 36hours. In some embodiments, such a treatment break ranges from about 24to about 48 hours. One of ordinary skill in the art can derive anappropriate dosing schedule based on common techniques and knowledge.

In embodiments, a compound of structure (I) or a pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt) and a second therapeuticagent are administered sequentially. In some embodiments, there is atreatment break between administering a compound of structure (I) or apharmaceutically acceptable salt thereof (e.g., a tartrate salt) andadministering the second therapeutic agent. In more specificembodiments, the treatment break is at least about 24 to about 48 hours.In particular embodiments, the treatment break is about 48 hours. Insome embodiments, the treatment break is at least about 120 hours.

4. Treatment of Ovarian Cancer

In embodiments, methods of the disclosure are useful for treating cancerin a subject. Such methods of treating a cancer (e.g., (i) arrest thecancer's development; (ii) cause regression of the cancer; or (iii)relieve the symptoms resulting from the cancer) include administering atartrate salt of a compound of structure (I), as described herein. Inembodiments, the cancer is ovarian clear cell carcinoma.

Additionally, in various embodiments, methods of the disclosure areuseful for treating ascites caused by a cancer in a subject.Accordingly, embodiments of the methods of treatment disclosed hereincomprise treating ascites in a subject in need thereof, the methodcomprising: administering a compound of structure (I), or apharmaceutically acceptable salt thereof (e.g., a tartrate salt),wherein the ascites is caused by a cancer. In some embodiments, thecancer is ovarian cancer.

In embodiments, the cancer is sensitive to a treatment. In embodiments,the cancer is partially sensitive to a treatment. In some embodiments,the cancer is refractory with regard to a treatment. In someembodiments, the cancer is recurrent. In embodiments, the cancer isresistant to a treatment. In embodiments, the cancer istreatment-resistant ovarian cancer.

In some embodiments, the treatment-resistant ovarian cancer is recurrenttreatment-resistant ovarian cancer. In some embodiments, the ovariancancer is drug-resistant ovarian cancer. In embodiments, the ovariancancer is resistant to a drug selected from the group consisting of ahormone therapeutic agent, a chemotherapeutic agent, animmunotherapeutic agent, or a cell growth factor and an agent to inhibitits receptor action.

In various embodiments, the drug is a hormone therapeutic agent.Examples of hormone therapeutic agents include fosfestrol,diethylstilbestrol, chlorotrianisene, medroxyprogesterone acetate,megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol,dienogest, asoprisnil, allylestrenol, gestrinone, nomegestrol, tadenan,mepartricin, raloxifene, ormeloxifene, levormeloxifene, antiestrogen(e.g., tamoxifen citrate, toremifene citrate, and the like), a pillformulation, mepitiostane, testololactone, aminoglutethimide, LH-RHderivatives (LH-RH agonist (e.g., goserelin acetate, buserelin,leuprorelin, and the like), LH-RH antagonist), droloxifene,epitiostanol, ethinyl estradiol sulfonate, aromatase inhibitors (e.g.,fadrozole hydrochloride, anastrozole, letrozole, exemestane, vorozole,formestane, and the like), antiandrogens (e.g., flutamide, bicalutamide,nilutamide, and the like), adrenocortical hormone-based agents (e.g.,dexamethasone, prednisolone, betamethasone, triamcinolone, and thelike), androgen synthesis inhibitors (e.g., abiraterone and the like),retinoid and an agent to retard the metabolism of retinoid (e.g.,liarozole and the like), and the like.

In various embodiments, the drug is a chemotherapeutic agent. In variousembodiments, the chemotherapeutic agent is an alkylating agent, anantimetabolite, an anti-cancer antibiotic, or a plant-derivedanti-cancer agent. In particular embodiments, the chemotherapeutic agentis an alkylating agent. Examples of alkylating agents include nitrogenmustard, nitrogen mustard N-oxide hydrochloride, chlorambucil,cyclophosphamide, ifosfamide, thiotepa, carboquone, improsulfantosilate, busulfan, nimustine hydrochloride, mitobronitol, melphalan,dacarbazine, ranimustine, estramustine sodium phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, ethoglucid,carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin,altretamine, ambamustine, dibrospidium hydrochloride, fotemustine,prednimustine, pumitepa, ribomustin, temozolomide, treosulfan,trophosphamide, zinostatin stimalamer, adozelesin, cystemustine,bizelesin and DDS formulations thereof, and the like. In specificembodiments, the alkylating agent is selected from the group consistingof carboplatin, cisplatin, miboplatin, nedaplatin, and oxaliplatin. Incertain embodiments, the alkylating agent is a cisplatin.

In embodiments, the chemotherapeutic agent is an antimetabolite.Examples of antimetabolites include mercaptopurine, 6-mercaptopurineriboside, thioinosine, methotrexate, pemetrexed, enocitabine,cytarabine, cytarabine ocfosphate, ancitabine hydrochloride, 5-FU basedagent (e.g., fluorouracil, tegafur, UFT, doxifluridine, carmofur,galocitabine, emitefur, capecitabine, and the like), aminopterin,nelzarabine, leucovorin calcium, Tabloid, butocin, calcium folinate,calcium levofolinate, cladribine, emitefur, fludarabine, gemcitabine,hydroxycarbamide, pentostatin, piritrexim, idoxuridine, mitoguazone,tiazofurin, ambamustine, bendamustine, and DDS formulations thereof, andthe like.

In embodiments, the chemotherapeutic agent is an anti-cancer antibiotic.Examples of anti-cancer antibiotics include actinomycin D, actinomycinC, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycinsulfate, peplomycin sulfate, daunorubicin hydrochloride, doxorubicinhydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride,epirubicin hydrochloride, neocarzinostatin, mithramycin, sarkomycin,carzinophilin, mitotane, zorubicin hydrochloride, mitoxantronehydrochloride, idarubicin hydrochloride, and DDS formulations thereof,and the like.

In embodiments, the chemotherapeutic agent is a plant-derivedanti-cancer agent. Examples of plant-derived anti-cancer agents includeetoposide, etoposide phosphate, vinblastine sulfate, vincristinesulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, DJ-927,vinorelbine, irinotecan, topotecan, and DDS formulations thereof, andthe like. Chemotherapeutic agents also include sobuzoxane.

In various embodiments, the drug is an immunotherapeutic agent. Examplesof immunotherapeutic agents include picibanil, krestin, schizophyllan,lentinan, ubenimex, interferon, interleukin, macrophagecolony-stimulating factor, granulocyte colony stimulating factor,erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum,levamisole, polysaccharide K, procodazole, anti-CTLA4 antibody, PD-1antibody, and Toll-like Receptors agonist (e.g., TLR7 agonist, TLR8agonist, TLR9 agonist, and the like).

In various embodiments, the drug is a cell growth factor. A cell growthfactor may be any substance as long as the substance promotes cellgrowth. Commonly, it includes a factor that is a peptide having amolecular weight of 20,000 or less and exhibits an effect at a lowconcentration by binding with the receptor. Specifically, epidermalgrowth factor (EGF) or substances having substantially the same activity(e.g., TGF-alpha and the like), insulin or substances havingsubstantially the same activity (e.g., insulin, insulin-like growthfactor (IGF)-1, IGF-2, and the like), fibroblast growth factor (FGF) orsubstances having substantially the same activity (e.g., acidic FGF,basic FGF, keratinocyte growth factor (KGK), FGF-10, and the like), andother cell growth factors (e.g., colony stimulating factor (CSF),erythropoietin (EPO), interleukin-2 (IL-2), nerve growth factor (NGF),platelet-derived growth factor (PDGF), transforming growth factor beta(TGF-beta), hepatocyte growth factor (HGF), vascular endothelial growthfactor (VEGF), heregulin, angiopoietin, and the like).

In various embodiments, the subject being treated has been previouslytreated with the treatment to which the ovarian cancer is resistant. Inembodiments, the ovarian cancer is refractory with regard to thetreatment.

In embodiments, the subject being treated has been previously treatede.g., with a first-line therapy. In some embodiments, the subject'scancer is resistant to the first-line therapy (e.g., surgical resection,platinum based therapy, etc.). In some embodiments, the first-linetherapy is a platinum-based therapy. In certain embodiments, theplatinum-based therapy includes administering cisplatin to the subject.In various embodiments, the cancer is refractory with regard to thefirst-line therapy.

In various embodiments, effective amounts of a compound of structure(I), or a pharmaceutically acceptable salt thereof (e.g., a tartratesalt), can decrease the number of tumor cells, decrease the number ofmetastases, decrease tumor volume, induce apoptosis of cancer cells,induce cancer cell death, induce radio-sensitivity in cancer cells,inhibit angiogenesis near cancer cells, inhibit cancer cellproliferation, inhibit tumor growth, prevent metastasis, reduce thenumber of metastases, increase life expectancy, prolong a subject'slife, reduce cancer-associated pain, and/or reduce relapse orre-occurrence of the cancer following treatment.

In embodiments, effective amounts of a compound of structure (I), or apharmaceutically acceptable salt thereof (e.g., a tartrate salt), canslow or reverse weight gain, reduce swelling of the abdomen, reducedifficulty breathing, eliminate the need or decrease the frequency toreduce ascites volume by paracentesis or a similar procedure, increaseappetite, decrease abdominal pain, decrease bloating, decrease nausea,decrease vomiting, and/or decrease heartburn following treatment.

In some embodiments, administration of a compound of structure (I), or apharmaceutically acceptable salt thereof (e.g., a tartrate salt), to asubject results in complete remission. In some embodiments,administration of a compound of structure (I), or a pharmaceuticallyacceptable salt thereof (e.g., a tartrate salt), to a subject results inminimal residual disease (MRD; deposits of residual tumor<1 cm). In someembodiments, administration of a compound of structure (I), or apharmaceutically acceptable salt thereof (e.g., a tartrate salt), to asubject results in no residual disease (NRD; no detectable residualdeposits). In some embodiments, administration of a compound ofstructure (I), or a pharmaceutically acceptable salt thereof (e.g., atartrate salt), to a subject results in gross residual disease (GRD;deposits of residual tumor>1 cm), but with a statistically significantdecrease in overall tumor volume as compared to earlier measurements. Inparticular embodiments, administration of a compound of structure (I),or a pharmaceutically acceptable salt thereof (e.g., a tartrate salt),to a plurality of subjects results in an increase in one or more ofprogression free survival, complete remission rate, event free survival,and/or overall survival relative to an untreated plurality of subjects.In embodiments, administration of a compound of structure (I), or apharmaceutically acceptable salt thereof (e.g., a tartrate salt), to asubject results in reversing epithelial-to-mesenchymal transition (EMT).

The compound of structure (I) may be in free-acid or free-base form, orin a pharmaceutically acceptable salt form. In embodiments of themethods of treatment disclosed herein comprise administering a tartratesalt (e.g., di-tartrate salt) of the compound of structure (I). In someembodiments, the tartrate salt is a di-tartrate salt. In someembodiments, the compound of structure (I) is present as a prodrug.

5. Additonal Methods for Treating Solid Cancers

Provided herein are methods of treating a solid tumor in a subject,wherein the solid tumor has progressed on immunotherapy. In certainembodiments, the tumor has progressed after achieving a best documentedresponse of at least stable disease (i.e., SD, PR, or CR) followingimmunotherapy. In certain embodiments, the tumor has progressed afterachieving a best documented response of at least stable disease (i.e.,SD, PR, or CR) following at least 2 cycles (e.g., 8 weeks) ofimmunotherapy. In certain embodiments, the method comprisesadministering to the subject a compound of structure (I) (e.g., tartratesalt, e.g., Form A). In certain embodiments, the method comprisesadministering to the subject Form A of the compound of structure (I). Incertain embodiments, the subject continues treatment with their previousimmunotherapy in combination with a compound of structure (I) (e.g.,tartrate salt, e.g., Form A).

Also provided herein are methods of treating EGFR+ non-small cell lungcancer (NSCLC) in a subject. In certain embodiments, the subject hasdemonstrated progression following TKI treatment. In certainembodiments, the subject has demonstrated progression following a bestdocumented response of at least stable disease (i.e., SD, PR, or CR) on≤2 lines of oral TKIs. In certain embodiments, the subject continuestreatment with their TKI regimen in combination with a compound ofstructure (I) (e.g., tartrate salt, e.g., Form A). In certainembodiments, the method comprises administering to the subject acompound of structure (I) (e.g., tartrate salt). In certain embodiments,the method comprises administering to the subject Form A of the compoundof structure (I).

Also provided herein are methods of treating BRAF-, KRAS-, orNRAS-mutated colorectal carcinoma (CRC) in a subject. In certainembodiments, the subject is a patient for whom there is no standardtherapy remaining. In certain embodiments, the method comprisesadministering to the subject a compound of structure (I) (e.g., tartratesalt). In certain embodiments, the method comprises administering to thesubject Form A of the compound of structure (I).

Also provided herein are methods of treating persistent and/or recurrentovarian cancer in a subject. In certain embodiments, the cancer isplatinum refractory and/or platinum resistant. In certain embodiments,the cancer is platinum refractory and/or platinum resistant and thesubject has been administered any number of lines of prior therapy. Incertain embodiments, the method comprises administering to the subject acompound of structure (I) (e.g., tartrate salt). In certain embodiments,the method comprises administering to the subject Form A of the compoundof structure (I).

Also provided herein are methods of treating BRAF-mutated melanoma in asubject. In certain embodiments, the subject has progressed onimmunotherapy. In certain embodiments, the subject has progressed on acombination BRAF/MEK inhibitor. In certain embodiments, the methodcomprises administering to the subject a compound of structure (I)(e.g., tartrate salt). In certain embodiments, the method comprisesadministering to the subject Form A of the compound of structure (I).

The following embodiments are with respect to any of the methods oftreatment provided herein. In certain embodiments, the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A) is administeredorally. In certain embodiments, the compound of structure (I) (e.g.,tartrate salt, e.g., Form A) is administered orally once daily. Incertain embodiments, the compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered orally once daily for 21 days, followed by7 drug free days (i.e., 28 day cycle). In certain embodiments, the28-day cycle is repeated. In certain embodiments, the dose of thecompound of structure (I) (e.g., tartrate salt, e.g., Form A) isrecalculated at the beginning of each new treatment cycle to reflectchanges in the body surface area (BSA) that may have occurred during theprevious cycle. In certain embodiments, doses are only adjusted if thereis a ≥10% increase or decrease in body weight compared to baseline. Incertain embodiments, dosing is repeated every cycle in the absence ofdisease progression or unacceptable toxicity. In certain embodiments,the compound of structure (I) (e.g., tartrate salt, e.g., Form A) istaken in the morning after an overnight fast with up to 200 mL or 7fluid ounces of water at least 1 hour before ingesting any food or othermedications.

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is administered daily at a dose from 1 mg/m² to 65mg/m², inclusive. In certain embodiments, the compound of structure (I)(e.g., tartrate salt, e.g., Form A) is administered daily at a dose ofabout 1.5 mg/m², 3.0 mg/m², 6.0 mg/m², 9.0 mg/m², 12.0 mg/m², 16.0mg/m², 21.0 mg/m², 28.0 mg/m², 37.0 mg/m², 49.0 mg/m², or 65.0 mg/m². Incertain embodiments, the compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered daily at a dose greater than 65.0 mg/m².

In certain embodiments, compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered at a starting dose of 1.5 mg/m². Incertain embodiments, compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered at a starting dose of 1.5 mg/m² for 21 outof 28 days in a 28 day dose cycle. In certain embodiments, the dose ofcompound of structure (I) (e.g., tartrate salt, e.g., Form A) isincreased between dose cycles. In certain embodiments, the dose ofcompound of structure (I) (e.g., tartrate salt, e.g., Form A) isincreased 30-100% between cycles.

In certain embodiments, compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered at a starting dose of 1.5 mg/m² for 21 outof 28 days in 3-subject cohorts using a standard 3+3 design. Forinstance, in certain embodiments, once the first subject in each cohorthas completed 14 days of dosing with no DLTs, the second and thirdsubjects are enrolled simultaneously at the same dose. In certainembodiments, once the last subject enrolled has completed Day 28 withoutobservation of a DLT and the next higher the compound of structure (I)dose level has not yet been studied, the dose is increased following amodified Fibonacci dose escalation scheme in a new 3-subject cohortaccording to the dose levels provided in Table 20.

In certain embodiments, If a DLT is observed in 1 of 3 subjects at agiven dose level, up to 3 additional subjects are enrolled and treatedat that dose level. In certain embodiments, when up to 3 additionalsubjects are added to a given dose level, if only 1 out of those 6subjects experiences a DLT, the dose is increased to the next doselevel. In certain embodiments, if ≥2 out of 3-6 subjects at a dose levelexperience DLTs, the dose is decreased to the previous (lower) doselevel and 3 additional subjects will be enrolled at that dose level.

In certain embodiments, if 0 or 1 subject in any of the 6 subjectsexperience a DLT, but the next higher dose level has already beenstudied, then the current dose is declared the MTD. In certainembodiments, the MTD is defined as the dose at which ≤1 of 6 subjectsexperience a DLT during Cycle 1 with the next higher dose having atleast 2 of 3-6 subjects experiencing a DLT during Cycle 1.

In certain embodiments, compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered as a flat dose (i.e., instead of accordingto BSA). In certain embodiments, the flat dose is the MTD.

In certain embodiments, the dosage of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt, e.g.,Form A) is about 1-37 mg/m² (e.g., 1-25 mg/m²) or about 1-75 mg (e.g.,1-50 mg) daily.

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is supplied in oral form as a powder in hard gelatincapsules. In certain embodiments, the compound of structure (I) (e.g.,tartrate salt, e.g., Form A) capsules are formulated in 1-mg, 4-mg,16-mg, 25-mg, or 100 mg strengths. In certain embodiments, the compoundof structure (I) (e.g., tartrate salt, e.g., Form A) are packaged intoround high-density polyethylene bottles with polyester coils asheadspace fillers. In certain embodiments, the bottles are thenheat-sealed, fitted with child-resistant caps, and placed in low-densitypolyethylene bags as secondary packaging.

6. Additional Methods for Treating CLL/SLL

Provided herein are methods of treating chronic lymphocytic leukemia(CLL) and/or small lymphocytic lymphoma (SLL) in a subject. In certainembodiments, the subject is intolerant to, or has shown progressivedisease on B-cell receptor antagonists and/or BCL-2 antagonists. Incertain embodiments, the subject is intolerant to, or has shownprogressive disease on other investigational treatments for CLL/SLL. Incertain embodiments, the subject has shown progression of disease onibrutinib. In certain embodiments, the method comprises administering tothe subject a compound of structure (I) (e.g., tartrate salt, e.g., FormA). In certain embodiments, the method comprises administering to thesubject Form A of the compound of structure (I).

The following embodiments are with respect to any of the methods oftreatment provided herein. In certain embodiments, the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A) is administeredorally. In certain embodiments, the compound of structure (I) (e.g.,tartrate salt, e.g., Form A) is administered orally once daily. Incertain embodiments, the compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered orally once daily for a 28 day cycle. Incertain embodiments, dosing is repeated for one or more 28-day cycles(e.g., in absence of disease progression or unacceptable toxicity). Incertain embodiments, the compound of structure (I) (e.g., tartrate salt,e.g., Form A) is taken in the morning after an overnight fast with up to200 mL or 7 fluid ounces of water at least 1 hour before ingesting anyfood or other medications. In certain embodiments, ibrutinib isadministered orally once daily in combination with the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A).

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is administered daily at a flat dose of 20 mg to 100mg, inclusive. In certain embodiments, the compound of structure (I)(e.g., tartrate salt, e.g., Form A) is administered daily at a flat doseof about 20, 25, 33, 45, 50, 58, 75, or 100 mg. In certain embodiments,the compound of structure (I) (e.g., tartrate salt, e.g., Form A) isadministered daily at a flat dose of greater than 100 mg

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is administered as a monotherapy. In certainembodiments, the compound of structure (I) (e.g., tartrate salt, e.g.,Form A) is administered at a 25 mg flat dose as a monotherapy. Incertain embodiments, the compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered orally once daily at a 25 mg flat dose for28 days. In certain embodiments, the 28-day cycle is repeated one ormore times. In certain embodiments, the subject continues to receive thecompound of structure (I) (e.g., tartrate salt, e.g., Form A) in 28-daycycles at the same dose given until they experience unacceptabletoxicity or unequivocal disease progression. In certain embodiments, thedose of the compound of structure (I) (e.g., tartrate salt, e.g., FormA) is increased between cycles. In certain embodiments, the dose isincreased by about 25-36% between cycles. In certain embodiments, thedose is increased based on the dose escalation levels in Table 22.

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is administered in combination with ibrutinib. Incertain embodiments, the compound of structure (I) (e.g., tartrate salt,e.g., Form A) is administered daily at a 20-mg flat dose in combinationwith ibrutinib. In certain embodiments, the compound of structure (I)(e.g., tartrate salt, e.g., Form A) is administered orally once daily ata 20-mg flat dose for 28 days in combination with ibrutinib. In certainembodiments, ibrutinib is administered at the same dose that they werereceiving immediately prior to beginning treatment with the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A). In certainembodiments, a subject continues with the combination of ibrutinib andthe compound of structure (I) (e.g., tartrate salt, e.g., Form A) for atleast 3 months after the start of treatment with the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A). In certainembodiments, ater that time, subjects either continue with combinationtherapy or discontinue ibrutinib and continue with the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A) monotherapy. Incertain embodiments, ibrutinib administration is stopped andreinitiated. In certain embodiments, subject continues to receive thecompound of structure (I) (e.g., tartrate salt, e.g., Form A) in 28-daycycles at the same dose until they experience unacceptable toxicity orunequivocal disease progression. In certain embodiments, the 28-daycycle is repeated one or more times. In certain embodiments, the subjectcontinues to receive the compound of structure (I) (e.g., tartrate salt,e.g., Form A) in 28-day cycles at the same dose given until theyexperience unacceptable toxicity or unequivocal disease progression.

In certain embodiments, the dose of the compound of structure (I) (e.g.,tartrate salt, e.g., Form A) is increased between 28 day cycles. Incertain embodiments, the dose is increased by about 25-36% betweencycles. In certain embodiments, the dose is increased based on the doseescalation levels in Table 22.

In certain embodiments, as described above, the dose of the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A) is increased betweencycles. In certain embodiments, dose escalation of the compound ofstructure (I) (e.g., tartrate salt, e.g., Form A) follows a standard 3+3design with sequential cohorts of 3 subjects treated with incrementallyhigher doses of the compound of structure (I) (e.g., tartrate salt,e.g., Form A) until a DLT is observed and the MTD is established. Incertain embodiments, once the first subject at a dose level is enrolled,the second and third subjects are enrolled after 3 weeks if the initialsubject has not experienced a DLT or any unacceptable toxicity.

In certain embodiments, if 1 of 3 subjects in a cohort experiences aDLT, up to 3 additional subjects are treated at that dose level. Incertain embodiments, if no additional DLTs are observed in the expanded3- to 6 subject cohort within 28 days after the last subject was firstdosed, the dose is escalated in a new cohort of 3 subjects. In certainembodiments, if 2 or more of 3 to 6 subjects at a given dose levelexperience a DLT during the first cycle, then the MTD is exceeded and upto a total of 6 subjects will be treated at the previous lower doselevel. In certain embodiments, if 0 or 1 of 6 subjects experiences a DLTat this previous lower dose level, this dose is declared the MTD. TheMTD is defined as the dose at which ≤1 of 6 subjects experience a DLTduring Cycle 1 with the next higher dose having at least 2 of 3 to 6subjects experiencing a DLT during Cycle 1. In certain embodiments, oncethe MTD or preliminary recommended phase 2 dose (RP2D) is identified, anexpansion cohort of up to six subjects is enrolled in each subjectgroup. In certain embodiments, the MTD is the RP2D.

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is administered at the RP2D orally once daily for 28days. In certain embodiments, dosing with the compound of structure (I)(e.g., tartrate salt, e.g., Form A) may continue until a subjectexperiences unacceptable toxicity or unequivocal disease progression.

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is administered at the RP2D orally once daily for 28days in combination with ibrutinib. In certain embodiments, the subjectis administered ibrutinib at the same dose that they were receivingimmediately prior to starting treatment with the compound of structure(I) (e.g., tartrate salt, e.g., Form A).

In certain embodiments, the dosage of the compound of structure (I) orpharmaceutically acceptable salt thereof (e.g., a tartrate salt, e.g.,Form A) is about 1-37 mg/m² (e.g., 1-25 mg/m²) or about 1-75 mg (e.g.,1-50 mg) daily. In certain embodiments, the dosage of the compound ofstructure (I) or pharmaceutically acceptable salt thereof (e.g., atartrate salt, e.g., Form A) is about 1-37 mg/m² (e.g., 1-25 mg/m²) orabout 1-75 mg (e.g., 1-50 mg) daily in combination with ibrutinib.

In certain embodiments, the compound of structure (I) (e.g., tartratesalt, e.g., Form A) is supplied in oral form as a powder in hard gelatincapsules. In certain embodiments, the compound of structure (I) (e.g.,tartrate salt, e.g., Form A) capsules are formulated in 1-mg, 4-mg,16-mg, 25-mg, and 100 mg strengths. In certain embodiments, the compoundof structure (I) (e.g., tartrate salt, e.g., Form A) are packaged intoround high-density polyethylene bottles with polyester coils asheadspace fillers. In certain embodiments, the bottles are thenheat-sealed, fitted with child-resistant caps, and placed in low-densitypolyethylene bags as secondary packaging.

7. Additional therapeutic uses of the compound of structure (I) (e.g.,freebase or a pharmaceutically acceptable salt thereof, such as tartratesalt, e.g., Form A) include treatment of melanoma (e.g., BRAF-melanomaand/or metastatic melanoma), either alone or in combination with one ormore checkpoint inhibitors, for example, checkpoint inhibitors of PD-1,PD-L1 or CTLA-4). Any of such checkpoint inhibitors can be co-used withthe compound of structure (I) in melanoma treatment. In some examples,the checkpoint inhibitor can be a PD-1 inhibitor such as pembrolizumab.Any of the treatment conditions (e.g., dosage, dosing schedule,administration route, etc.) as disclosed herein can be applied inmelanoma treatment as disclosed herein.

8. Additional therapeutic uses of the compound of structure (I) (e.g.,freebase or a pharmaceutically acceptable salt thereof, such as tartratesalt, e.g., Form A) also include treatment of brain tumor (e.g., GBM),either alone or in combination with one or more checkpoint inhibitors,for example, checkpoint inhibitors of PD-1, PD-L1 or CTLA-4). Any ofsuch checkpoint inhibitors can be co-used with the compound of structure(I) in melanoma treatment. In some examples, the checkpoint inhibitorcan be a PD-1 inhibitor such as pembrolizumab. Any of the treatmentconditions (e.g., dosage, dosing schedule, administration route, etc.)as disclosed herein can be applied in melanoma treatment as disclosedherein.

E. Methods of Synthesis

1. Synthesis of Tartrate Salts of the Compound of Structure (I)

It will be appreciated by those skilled in the art that the processesand reactions for preparing the compounds described herein (includingthe salts of the compound of structure (I)) may be modified inaccordance with standard techniques to include alternative reagentsand/or reaction conditions. For example, a reaction intermediate havinga halo substituent (i.e., F, Cl, Br, I) may alternatively employ asulfonate. Exemplary sulfonates are also referred to as “pseudohalides”that include, but are not limited to p-toluenesulfonate (OTs),methanesulfonate (OMs) or perfluoroalkylsulfonates (e.g., triflate).

Reducing agents include lithium aluminum hydride, nascent (atomic)hydrogen, hydrogen without or with a suitable catalyst, e.g., a Lindlarcatalyst, sodium or zinc amalgam (Na(Hg) or Zn(Hg)), sodium-lead alloy(Na+Pb), diborane, sodium borohydride, borane tetrahydrofuran,iron-based reducing agents (e.g., iron(II) sulfate), tin-based reducingagents (e.g., tin(II) chloride), sulfur dioxide, sulfite compounds,dithionates (e.g., Na₂S₂O₆), thiosulfates (e.g., Na₂S₂O₃), hydrazine,diisobutylaluminum hydride, oxalic acid, formic acid, ascorbic acid,reducing sugars, phosphites, hypophosphites, dithiothreitol (DTT),tris-2-carboxyethylphosphine hydrochloride (TCEP).

Accordingly, one embodiment (hereinafter “Step A”) provides a method forpreparing a compound of structure (I):

or a pharmaceutically acceptable salt thereof (e.g., a tartrate saltthereof), the method comprising reacting a compound having the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein

X is a leaving group;

Y is halo;

Z is halo or —NR¹(R²); and

R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl,

with a compound having the following structure:

wherein P is H or a protecting group, to obtain a compound having thefollowing structure:

In some embodiments, X is halo or sulfonate. In some specificembodiments, X is fluoro, chloro, bromo, or iodo. In some more specificembodiments, X is chloro.

In some of the foregoing embodiments, P is H. In certain embodiments, Pforms a methyl ester, a benzyl ester, a tert-butyl ester, a silyl ester,an ortho-ester or an oxazoline.

Another embodiment (hereinafter “Step B”) provides a method forpreparing a compound of structure (1):

or a pharmaceutically acceptable salt thereof (e.g., a tartrate saltthereof), the method comprising reacting a compound having the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein:

Y is halo;

Z is halo or —NR¹(R²); and

R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl,

with a reducing agent to obtain a compound having the followingstructure:

In some embodiments, the reducing agent is lithium aluminum hydride,diborane, sodium borohydride, borane, or combinations thereof. In somemore specific embodiments, the reducing agent is borane.

Yet another embodiment (hereinafter “Step C”) provides a method forpreparing a compound of structure (1):

or a pharmaceutically acceptable salt thereof (e.g., a tartrate saltthereof), the method comprising reacting a compound having the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein:

Y is halo;

Z is halo or —NR¹(R²); and

R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl,

with an activating agent to obtain a compound having the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein:

X′ is a leaving group.

In some embodiments, the activating agent comprises a sulfonyl chloridefunctional group. For example, in certain embodiments, the activatingcompound is thionyl chloride.

Still another embodiment (hereinafter “Step D”) provides a method forpreparing a compound of structure (I):

or a pharmaceutically acceptable salt thereof (e.g., a tartrate saltthereof), the method comprising reacting a compound having the followingstructure:

or a pharmaceutically acceptable salt thereof, wherein:

X′ is a leaving group;

Y is halo;

Z is halo or —NR¹(R²); and

R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl,

with N-methylpiperazine, or a salt thereof to obtain a compound havingthe following structure:

or a pharmaceutically acceptable salt thereof.

In some of the foregoing embodiments, X′ is halo or sulfonate. In someembodiments, X′ is fluoro, chloro, bromo, or iodo. In certainembodiments, X′ is chloro.

In some embodiments, Y is fluoro, chloro, bromo, or iodo. For example,in some specific embodiments, Y is chloro.

In some particular embodiments, Z is —NR¹(R²). In some more specificembodiments, R¹ is C₁-C₈ alkyl. In other related embodiments, R² isC₁-C₈ alkyl. In more specific embodiments, R¹ is methyl, ethyl, propylor isopropyl. For example, in certain embodiments, R¹ is methyl.Similarly, in some embodiments, R² is methyl, ethyl, propyl orisopropyl. For example, in certain embodiments, R² is methyl.

Another embodiment (hereinafter “Step E”) provides a method forpreparing a tartrate salt of a compound of structure (I):

the method comprising admixing the compound of structure (I) withtartaric acid.

In some embodiments, the method comprises adding tartaric acid to thecompound of structure (I). For example, in certain embodiments, themethod comprises adding L-(+)-tartaric acid to the compound of structure(I). In certain embodiments, the tartaric acid is added to the compoundof structure (I) in a ratio of about 1:1 to about 2:1, e.g., 1:1 or 2:1.In certain embodiments, the tartrate salt is characterized as having astoichiometry as described above.

Certain embodiments provide a method for preparing a compound ofstructure (I) or a pharmaceutically acceptable salt thereof (e.g., atartrate salt thereof) comprising Steps A and B, Steps A, B, and C,Steps A, B, C, and D, Steps A, B, C, D and E, Steps B and C, Steps B, Cand D, Steps B, C, D and E, Steps C and D, Steps C, D and E, or Steps Dand E. In certain embodiments, the method comprises Steps A and C, StepsA, C, and D, Steps A, C, D and E, Steps A, B, and D, Steps A, B, D andE, Steps A, B, C and E, Steps B and D, Steps B, D and E, Steps A and C,Steps A and D, Steps A and E, Steps B and E or Steps C and E.

It will also be appreciated by those skilled in the art that in theprocesses for preparing the compounds described herein the functionalgroups of intermediate compounds may need to be protected by suitableprotecting groups. Such functional groups include, but are not limitedto, hydroxy, amino, mercapto and carboxylic acid. Suitable protectinggroups for hydroxy include trialkylsilyl or diarylalkylsilyl (forexample, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),tetrahydropyranyl, benzyl, and the like. Suitable protecting groups foramino, amidino and guanidino include f-butoxycarbonyl,benzyloxycarbonyl, and the like. Suitable protecting groups for mercaptoinclude —C(O)—R″ (where R″ is alkyl, aryl or arylalkyl),p-methoxybenzyl, trityl and the like. Suitable protecting groups forcarboxylic acid include alkyl, aryl or arylalkyl esters. Protectinggroups are optionally added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green,T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compoundsdisclosed herein are included within the scope of embodiments of theinvention.

The following General Reaction Scheme illustrates an exemplary method offorming a tartrate salt of a compound of structure (I):

It is understood that one skilled in the art may be able to make thesesalts by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othersalts of a compound of structure (I) not specifically illustrated belowby using the appropriate starting components and modifying theparameters of the synthesis as needed. In general, starting componentsmay be obtained from sources such as Sigma Aldrich, Lancaster Synthesis,Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

Compounds and salts were analyzed using techniques known in the art, forexample, by x-ray powder diffraction (XRPD), dynamic vapor sorption(DVS), thermal gravimetric analysis (TGA), differential scanningcalorimetry (DSC), mass spectrometry and/or ¹H NMR. Synthetic proceduresare described in more detail below.

2. Syntheses of Crystalline Forms

Embodiments of the tartaric acid salt of a compound of structure (I) andpolymorphs thereof (e.g., Form A) can be prepared according to theGeneral Reaction Scheme below, wherein each occurrence of X is a halideor pseudohalide (e.g., triflate, nonaflate, mesylate, tosylate, etc.).Certain intermediates useful for preparation of a tartaric acid salt ofthe compound of structure (I) can be prepared according to methodsdescribed in WO 2012/135800, which is incorporated herein by referencein its entirety. As shown in the General Reaction Scheme, compounds ofstructure A1 can be purchased from commercial sources or preparedaccording to methods familiar to one of ordinary skill in the art,including those provided in the Examples (see, e.g., Example 6).Reaction of A1 with amine reagent A′ (which is prepared according toknown methods or purchased from commercial sources) yields A2. Phenylnitro compound A2 can then be converted to the aniline A3, which iscoupled with A4 to yield the pyrimidine containing product A5. Thepyrimidine containing A5 is then coupled to aniline compound A6 toafford A7. A7 can then be reduced as necessary (e.g., using BH₃.THF) andactivated (e.g., using thionyl chloride, Comins' reagent) to yieldcompound A8 (i.e., structure (I)). The compound A8 is then purified andconverted to the desired polymorph/salt by adding the appropriate acid(e.g., tartaric) under the appropriate conditions (e.g., heating coolingfollowing two re-slurry purification steps).

It should be noted that the General Reaction Scheme only depicts anexemplary method for preparation of a tartaric acid salt of a compoundof structure (I) and other methods are available, including methods forpreparation of a tartaric acid salt of compounds of structure (I) usingdifferent reagents, and/or different intermediates etc. Other specificembodiments provide methods for preparing the polymorphs from a compoundof structure (I). For example, one embodiment provides a method forpreparing a polymorph, the method comprising:

a) purifying a free base of a compound having the following structure(I):

thereby isolating a purified free base comprising less than about 30 mol% triethylamine;

b) dissolving the purified free base to form a recrystallizationsolution comprising the purified free base; and

c) treating the recrystallization solution with tartaric acid, therebyforming the polymorph.

In some embodiments, the purified free base comprises less than about 25mol % triethylamine, less than about 20 mol % triethylamine, less thanabout 15 mol % triethylamine, less than about 10 mol % triethylamine,less than about 5 mol % triethylamine, less than about 4 mol %triethylamine, less than about 3 mol % triethylamine, less than about 2mol % triethylamine, less than about 1 mol % triethylamine, or less thanabout 0.5 mol % triethylamine.

In some specific embodiments, the tartaric acid is L-(+)-tartaric acid.

In some specific embodiments, the tartaric acid is added at a molarratio ranging from about 2.5:1 to about 0.75:1 relative to the freebase. In more specific embodiments, the tartaric acid is added at amolar ratio ranging from about 2.3:1 to about 0.9:1 relative to the freebase. In more specific embodiments, the tartaric acid is added at amolar ratio ranging from about 2.2:1 to about 1.5:1 relative to the freebase. In certain embodiments, the tartaric acid is added at a molarratio of about 2:1 relative to the free base.

In some embodiments, the purifying comprises a first purificationcomprising contacting the free base with a first solvent thereby forminga first suspension. In some embodiments, the first solvent is an organicsolvent. In some embodiments, the first solvent is an alcohol (e.g.,methanol, ethanol, isopropyl alcohol, n-propyl alcohol, butanol, and thelike). In some specific embodiments, the first solvent is ethanol.

In certain embodiments, the first purification further comprises heatingthe first suspension at a temperature of at least about 50° C. In somemore specific embodiments, the first purification further comprisesheating the first suspension at a temperature of at least about 65° C.In some more specific embodiments, the first purification furthercomprises heating the first suspension at a temperature of at leastabout 45° C. 55° C. 57° C. 60° C. 62° C. 67° C. 70° C. 72° C., or 75° C.

In some embodiments, the first purification further comprises removingthe first solvent, thereby forming a solid product.

In some embodiments, the method further comprises a second purificationcomprising contacting the solid product with a purifying mixture,thereby forming a second suspension. In some embodiments, the purifyingmixture comprises a second solvent, a third solvent, and a purifyingreagent. In some more specific embodiments, the second solvent is anorganic solvent. In some embodiments, the second solvent is an alcohol.In some embodiments, the second solvent is ethanol. In certainembodiments, the third solvent is an organic solvent. In more specificembodiments, the third solvent is chloroform. In certain embodiments,the purifying reagent is activated charcoal.

In some more specific embodiments, the recrystallization solutionfurther comprises a recrystallization solvent. In more specificembodiments, the recrystallization solvent comprises an organic solvent.In some embodiments, the recrystallization solvent comprises an alcohol.In some embodiments, the recrystallization solvent comprises ethanol. Insome embodiments, the recrystallization solvent further comprisesanisole.

In certain embodiments, the dissolving further comprises heating therecrystallization solution at a temperature of at least about 50° C. Inmore specific embodiments, the heating is at a temperature of at leastabout 60° C. In still more specific embodiments, the heating is at atemperature of at least about 65° C. In still more specific embodiments,the heating is at a temperature of at least about 55° C. 57° C. 62° C.67° C. 70° C. 72° C., or 75° C.

In some embodiments, the method further comprises cooling therecrystallization solution at a temperature below about 25° C. (e.g.,below about 20° C. 15° C. 10° C. or 0° C.).

In some more specific embodiments, the method further comprises coolingthe recrystallization solution at a temperature below about 20° C. Insome more specific embodiments, the method further comprises isolatingthe polymorph as a solid (e.g., a crystalline solid).

In more specific embodiments, the crystalline form has a ratio rangingfrom about 2.25:1 to about 1.75:1 of tartaric acid to compound ofstructure (I). In some embodiments, the crystalline form has a ratioranging from about 2.1:1 to about 1.9:1 of tartaric acid to compound ofstructure (I). In some embodiments, the polymorph has a ratio of about2:1 of tartaric acid to compound of structure (I). In certainembodiments, the polymorph is a L-(+)-tartaric acid salt. Certainembodiments provide a polymorph prepared according to the method of anyone of the foregoing embodiments.

3. Intermediates

In another aspect, the present disclosure provides a compound ofstructure (III):

or a pharmaceutically acceptable salt, or tautomer thereof, wherein:

-   -   Y is halo;    -   Z is halo or —NR¹(R²);    -   R¹ and R² are, each independently, hydrogen or C₁-C₈ alkyl;    -   R³ is halo or OR^(a);    -   R⁴ is hydrogen or oxo; and    -   R^(a) is hydrogen or C₁-C₈ alkyl.

In some embodiments, Y is chloro.

In some embodiments, Z is —NR¹(R²). In some particular embodiments R¹ isC₁-C₈ alkyl. In some particular embodiments R² is C₁-C₈ alkyl. In someembodiments, R¹ and R² are C₁-C₈ alkyl. For example, in someembodiments, R¹ is methyl. In another embodiment, R² is methyl. In yetanother embodiment, R¹ and R² are methyl.

In some related embodiments, R³ is OR^(a). In some embodiments R^(a) isH. In certain embodiments, R^(a) is methyl, ethyl or isopropyl. In someembodiments, R³ is halo. In some embodiments, R³ is chloro.

In some embodiments, R⁴ is hydrogen. In some other embodiments, R⁴ isoxo.

In certain specific embodiments, the compound has one of the followingstructures:

F. Metabolites

1. Compounds

The compound of structure (I) undergoes metabolic conversion into anumber of species. See, e.g., FIG. 100. Various embodiments of suchcompounds are provided as structure (IV), detailed below. Also providedare methods for preparing the same. Some embodiments include compoundintermediates useful at least for preparing compounds of structure (IV),all of which are considered to be included as a part of the presentdisclosure. One embodiment provides a compound having the followingstructure (IV):

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof,

wherein:

-   -   A represents a 6-membered aromatic ring or a 6-membered        carbocyclic ring;    -   R^(1a) and R^(1b) are each independently H, C₁-C₆ alkyl, or —OH;    -   R² and R³ are each independently H, C₁-C₆ alkyl, or halo;    -   R⁴ is H, C₁-C₆ alkyl, or —OH;    -   R^(5a) and R^(5b) are each independently H, C₁-C₆ alkyl, or        halo;    -   R^(6a), R^(6b), R^(6C) and R^(6d) are each independently absent        or —O⁻;    -   R⁷ is H, C₁-C₆ alkyl, —OH or absent;    -   R⁸ is absent or has the following structure:

-   -   R⁹ is absent or alkenyl, provided that at least one of R⁸ or R⁹        is present;    -   R¹⁰ is H or C₁-C₆ alkyl; and    -   represents a double or single bond; and    -   all valencies are satisfied;    -   provided that if R^(1a) and R^(1b) are both methyl, then:    -   a. at least one of R^(6a), R^(6b), R^(6c), R^(6d) and R⁹ is —O⁻;    -   b. R⁷ is C₁-C₆ alkyl, —OH or absent; and/or    -   c. R¹⁰ is H.

In some embodiments, the compound has one of the following structures(V) or (VI):

In some more specific embodiments, the compound has one of the followingstructures (IIa) or (IIIa):

In more specific embodiments, at least one of R^(1a) or R^(1b) is H. Insome embodiments, at least one of R^(1a) or R^(1b) is C₁-C₆ alkyl. Inmore specific embodiments, at least one of R^(1a) or R^(1b) is methyl.

In other embodiments, R^(1a) and R^(1b) are both H. In still otherembodiments, R^(1a) and R^(1b) are both C₁-C₆ alkyl, for example, R^(1a)and R^(1b) are both methyl.

In certain embodiments, R² is H. In related embodiments, R³ is H. Inother embodiments, R⁴ is H. In another embodiment, R⁴ is absent.

In some embodiments, R^(5a) is halo, for example, F, Cl, Br or I. Insome embodiments, R^(5a) is Cl. In certain related embodiments, R^(5b)is H.

In other specific embodiments, at least one of R^(6a), R^(6b), R^(6c)and R^(6d) are —O⁻, for example, R^(6a) is —O⁻, R^(6b) is —O⁻, R^(6c) is—O⁻ or R^(6d) is —O⁻. In some embodiments, R^(6d) is —O⁻.

In other specific embodiments, R⁷ is H. In another embodiment, R⁷ isabsent.

In certain embodiments, R¹⁰ is H. In another embodiment, R¹⁰ is C₁-C₆alkyl, for example, R¹⁰ is methyl, ethyl or propyl. In some embodiments,R¹⁰ is methyl.

In some specific embodiments, R⁸ has one of the following structures:

In one particular embodiment, R⁸ has the following structure:

In one particular embodiment, R⁸ has the following structure:

In one particular embodiment, R⁸ has the following structure:

In one embodiment, R⁹ is absent.

In another embodiment of structure (IV), the compound has one of thefollowing structures (VII) or (VIII):

In yet another embodiment of structure (IV), the compound has one of thefollowing structures (VIIIa), (VIIIb) or (VIIIc):

Another embodiment provides a compound having the following structure:

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof.

A different embodiment provides compound having the following structure:

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof.

Yet another embodiment provides a compound having the followingstructure:

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof,

An additional embodiment provides compound having the followingstructure:

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof.

Yet another embodiment affords compound having the following structure:

or a stereoisomer, pharmaceutically acceptable salt, tautomer or prodrugthereof.

In certain embodiments of the compound of structure (IV), the compoundis isolated using chemical techniques known in the art (e.g., columnpurification, distillation, recrystallization, etc.). Accordingly, incertain embodiments, the compound of structure (IV) is isolated. In someembodiments, the compound of structure (IV) is purified. In someembodiments, the compound of structure (IV) is isolated and purified.

Some embodiments provide a composition consisting essentially of acompound of structure (IV), that is, a composition that containssubstantially no impurities which have an adverse effect on compositionas it relates to inhibition of kinase activity as determined using invitro assays.

In some embodiments, the compound is selected from Table 7, andpharmaceutically acceptable salts thereof.

TABLE 7 Exemplary Compounds of Structure (IV) m/z No. Structure [M + H]⁺¹HNMR M2

488.2 FIG. 101A M3

502.2 FIG. 101B M4

502.2 FIG. 101C M5

416.1 M6

518.2 FIG. 101D M7

532.2 FIG. 101E

Table 7A summarizes AXL inhibition and AUC data for representativemetabolites of structure (IV).

TABLE 7A Metabolite Date % after 2 hrs % in plasma % in plasma exposureat day 21 at at day 21 at AXL IC50 to human 16 mg/M2 dose 21 mg/M2 doseCmpd (nM) hepatocytes (FIG. 102A) (FIG. 102B) (I) 4.6 55%  38% 17% M1 na1% not detected not detected M2 8.5 2%  8% 17% M3 8.7 13%  40% 44% M49.3 8% 10% 14% M5 na 2% not detected not detected M6 15.7  3%  3%  7% M723.4  15%  not detected not detected

Additionally, the foregoing embodiments include a compound of structure(IV) as a pharmaceutically acceptable salt, for example, an acidaddition salt or a base addition salt. In more specific embodiments, theacid addition salt is a hydrochloric salt, a fumaric salt, or a tartratesalt. In certain embodiments, the acid addition salt is a tartrate salt.

2. Methods

Certain methods disclosed herein serve to select a regimen of treatmentfor a subject in need thereof. That is, this disclosure provides methodsfor selecting treatment regimens as well as methods of treatmentthemselves. Additionally, certain embodiments provide a method forselecting treatment regimens and methods of treatment based on ametabolic profile. Other embodiments provide a method for selecting atreatment regimen and for treating cancer in a subject based on thesubject having a predetermined genetic profile.

Embodiments provided herein include methods for selecting a treatmentregimen for a subject based on the subject's genetic profile. Suchgenetic profiles may be produced in any suitable manner (e.g.,microarrays, reverse transcription polymerase chain reaction (RT-PCR),etc.)

In some embodiments, the genetic profile comprises a polymorphism in agene encoding a cytochrome P450 enzyme. The term “gene” can include notonly coding sequences but also regulatory regions such as promoters,enhancers, and termination regions. The term further can include allintrons and other DNA sequences spliced from the mRNA transcript, alongwith variants resulting from alternative splice sites. Gene sequencesencoding the particular protein can be DNA or RNA that directs theexpression of the particular protein. These nucleic acid sequences maybe a DNA strand sequence that is transcribed into RNA or an RNA sequencethat is translated into the particular protein. The nucleic acidsequences include both the full-length nucleic acid sequences as well asnon-full-length sequences derived from the full-length protein.

In some embodiments, the particular protein is an enzyme, for example acytochrome P450 enzyme. Cytochrome P450 (CYP) enzymes are a major sourceof variability in drug pharmacokinetics and response. In someembodiments the cytochrome P450 enzyme is in the CYP1, CYP2 or CYP3families. In embodiments, the cytochrome P450 enzyme is CYP1A2, CYP2A6,CYP2C8, CYP2C9, CYP3A43, CYP3A5, CYP4A11, CYP4A22, CYP4B1, CYP4F2,CYP5A1, CYP11A1, CYP11B1, CYP11B2, CYP1A1, CYP1A2, CYP1B1, CYP2A6,CYP2A7, CYP2B6, CYP2C18, CYP2C19, CYP2C19, CYP2C9, CYP2D6, CYP2D6,CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A4, CYP3A5,CYP3A7, CYP3A7, CYP4F11, CYP4F12, CYP4F22, CYP4F3, CYP4F8, CYP4V2,CYP4×1, CYP7A1, CYP7B1, CYP8B1, or CYPF22. In some embodiments, thecytochrome P450 enzyme is CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6,CYP2A6, CYP3A5, or CYP3A7. In some embodiments, the cytochrome P450enzyme is CYP3A4, CYP2C9, CYP2C8, CYP2E1, CYP1A2, CYP2A6, CYP2D6,CYP2B6, CYP2C19, CYP3A5, CYP2J2, CYP1A1, or CYP1B1. In some embodiments,the cytochrome P450 enzyme includes CYP2D6, CYP2C19, CYP2C9, CYP2B6,CYP3A5 or CYP2A6. In some embodiments, the cytochrome P450 enzyme isCYP1A1, CYP1A2, CYP2C8, CYP2E1, CYP2J2, or CYP3A4. In some specificembodiments, the cytochrome P450 enzyme is CYP2C19. In variousembodiments, the genetic profile comprises information regardingmultiple cytochrome P450 enzymes. In some embodiments, the polymorphismis a single nucleotide polymorphism.

In embodiments, the polymorphism results in:

i. an expression level of the cytochrome P450 enzyme that is differentthan a baseline expression level by at least 10%; and/or

ii. an activity of the cytochrome P450 enzyme that is different than abaseline activity by at least 10%.

Expression levels may be measured using any suitable techniques, such asenzyme-linked immunoassays (ELISA), mass spectrometry (MS), real-timequantitative PCR (RT-qPCR), flow cytometry, nucleic acid (i.e., DNA,RNA, etc.) sequencing, amino acid (i.e., peptide, protein, etc.)sequencing, molecular cytogenetics fluorescence in situ hybridization(FISH), and the like.

A baseline level can be derived from a population. A “population” is anygrouping of subjects or samples of like specified characteristics. Thegrouping could be according to, for example, clinical parameters,clinical assessments, therapeutic regimens, disease status, severity ofcondition, etc.

In some embodiments, the population is selected randomly. In someembodiments, the population is a group comprising about 2, about 5,about 10, about 25, about 50, about 75, or about 100 subjects. In someembodiments, the population is a group comprising about 200, about 300,about 500, about 1,000, about 1,500, about 2,000, about 3,000, about5,000, or about 10,000 subjects. In some embodiments, the population isa group comprising less than about 10,000 subjects. In otherembodiments, the population is a group comprising greater than about10,000 subjects.

In some embodiments, the population is a group that does not havecancer. In embodiments, the population does not have a hematologiccancer. In some embodiments, the population does not have a solid tumorcancer. In some embodiments, the population does not have liver cancer.

In some embodiments, the population is a group that has cancer. Inembodiments, the population has a hematologic cancer. In someembodiments, the population does has a solid tumor cancer. In someembodiments, the population has liver cancer. In some embodiments, thepopulation has a same type of cancer.

In some of the foregoing embodiments, the population is non-responsiveto an AXL kinase inhibitor. In some embodiments, the population isrefractory after treatment with an AXL kinase inhibitor. In someembodiments, the population is intolerant of treatment with an AXLkinase inhibitor. In some embodiments, the AXL kinase inhibitor is thecompound of structure (I), or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt). In some embodiments, the AXL kinase inhibitoris a compound of structure (IV) or a pharmaceutically acceptable saltthereof.

In various embodiments, an expression level of the cytochrome P450enzyme is greater than a baseline expression level. In embodiments, theexpression level of the cytochrome P450 enzyme is at least about 10%greater than the baseline expression level. In some embodiments, theexpression level is at least about 1% greater, at least about 2%greater, at least about 3% greater, at least about 4% greater, at leastabout 5% greater, at least about 7% greater, at least about 12% greater,at least about 15% greater, at least about 17% greater, at least about20% greater, at least about 22% greater, at least about 25% greater, atleast about 27% greater, at least about 30% greater, at least about 32%greater, at least about 35% greater, at least about 37% greater, atleast about 40% greater, at least about 45% greater, at least about 50%greater, at least about 75% greater, or at least about 90% greater. Incertain embodiments, a cytochrome P450 gene is up-regulated.“Up-regulation” or “up-regulated” refers to an increase in the presenceof a protein and/or an increase in the expression of its gene.

In some embodiments, an expression level of the cytochrome P450 enzymeis less than a baseline expression level. In embodiments, the expressionlevel of the cytochrome P450 enzyme is at least 10% less than thebaseline expression level. In some embodiments, the expression level isat least about 1% less, at least about 2% less, at least about 3% less,at least about 4% less, at least about 5% less, at least about 10% less,at least about 12% less, at least about 15% less, at least about 17%less, at least about 20% less, at least about 22% less, at least about25% less, at least about 27% less, at least about 30% less, at leastabout 32% less, at least about 35% less, at least about 37% less, atleast about 40% less, at least about 45% less, at least about 50% less,at least about 75% less, or at least about 90% less. In certainembodiments, a cytochrome P450 gene is down-regulated. “Down-regulation”or “down-regulated” refers to a decrease in the presence of a proteinand/or a decrease in the expression of its gene.

Measurement of expression of the markers can be determined at theprotein or nucleic acid level using any method known in the art. In someembodiments, a marker is detected by contacting a sample with reagents(e.g., antibodies or nucleic acid primers), generating complexes ofreagent and marker(s), and detecting the complexes. Antibodies can beconjugated to a solid support suitable for a diagnostic assay inaccordance with known techniques, such as passive binding. Antibodiescan be conjugated to cell surface antigens for a diagnostic assay inaccordance with known techniques, such as flow cytometry, includingmulti-color flow cytometry. Antibodies can be conjugated to detectablelabels or groups such as radiolabels, enzyme labels, and fluorescentlabels in accordance with known techniques.

Examples of suitable immunoassays include immunoblotting,immunoprecipitation, immunofluorescence, chemiluminescence,electro-chemiluminescence (ECL), and ELISA. Up- or down-regulation ofmarkers also can be detected using, for example, cDNA arrays, clonehybridization, differential display, differential screening, FRETdetection, liquid microarrays, PCR, RT-PCR, Sanger sequencing,mass-parallel (next-generation) sequencing, molecular beacons,microelectric arrays, oligonucleotide arrays, polynucleotide arrays,serial analysis of gene expression (SAGE), and/or subtractivehybridization.

Expression may be determined in a sample collected from a subject priorto treatment. In such embodiments, expression levels may be used topredict responsiveness to a particular treatment. In some embodiments,expression levels may be used, at least in part, to determine atreatment administered to a subject. In some embodiments, a sample maybe collected after a dose of a treatment is administered to a subject.In specific embodiments, a sample is collected on day 1 of a first cycleof treatment, pre-dose, two hours after dosing, six hours after dosing,and 24 hours after dosing. In another specific embodiment, a sample isalso collected on day 8 of the first cycle of treatment, pre-dose. Inanother specific embodiment, a sample is also collected on day 1 of thesecond cycle of treatment, pre-dose. In further specific embodiment, asample is also collected on day 1 of any additional cycles of treatment(e.g., third, fourth, fifth, etc.), pre-dose. In another specificembodiment, a sample is also collected after treatment is completed.

Up- or down-regulation can be assessed by comparing a value to arelevant reference level. For example, the quantity of one or moremarkers can be indicated as a value, which can be derived, e.g., bymeasuring level(s) of the marker(s) in the sample by an assay performed.In the broadest sense, the value may be qualitative or quantitative.Where detection is qualitative, the systems and methods provide areading or evaluation, e.g., assessment, of whether or not the marker ispresent in the sample being assayed. In yet other embodiments, thesystems and methods provide a quantitative detection of whether themarker is present in the sample being assayed, i.e., an evaluation orassessment of the actual amount or relative abundance of the marker inthe sample being assayed. In such embodiments, the quantitativedetection may be absolute or, if the method is a method of detecting twoor more different markers in a sample, relative. Accordingly, the term“quantifying” when used in the context of quantifying a marker in asample can refer to absolute or to relative quantification. Absolutequantification can be accomplished by inclusion of knownconcentration(s) of one or more control markers and referencing, e.g.,normalizing, the detected level of the marker with the known controlmarkers (e.g., through generation of a standard curve). Alternatively,relative quantification can be accomplished by comparison of detectedlevels or amounts between two or more different markers to provide arelative quantification of each of the two or more markers, e.g.,relative to each other.

In various embodiments an activity of the cytochrome P450 enzyme isgreater than a baseline activity. The function of a protein can beassayed by a relevant activity assay. Exemplary activity assays includebinding assays, enzyme activity assays including, for example, proteaseassays, kinase assays, phosphatase assays, reductase assays, etc.

In some embodiments, the activity of one or more cytochrome P450 enzymesis at least about 10% greater than the baseline activity. In someembodiments, the activity is at least about 1% greater, at least about2% greater, at least about 3% greater, at least about 4% greater, atleast about 5% greater, at least about 7% greater, at least about 12%greater, at least about 15% greater, at least about 17% greater, atleast about 20% greater, at least about 22% greater, at least about 25%greater, at least about 27% greater, at least about 30% greater, atleast about 32% greater, at least about 35% greater, at least about 37%greater, at least about 40% greater, at least about 45% greater, atleast about 50% greater, at least about 75% greater, or at least about90% greater.

In some embodiments, an activity of the cytochrome P450 enzyme is lessthan a baseline activity. In embodiments, the activity of the cytochromeP450 enzyme is at least about 10% less than the baseline activity. Insome embodiments, the activity is at least about 1% less, at least about2% less, at least about 3% less, at least about 4% greater, at leastabout 5% greater, at least about 7% less, at least about 12% less, atleast about 15% less, at least about 17% less, at least about 20% less,at least about 22% less, at least about 25% less, at least about 27%less, at least about 30% less, at least about 32% less, at least about35% less, at least about 37% less, at least about 40% less, at leastabout 45% less, at least about 50% less, at least about 75% less, or atleast about 90% less.

Accordingly, embodiments of the present disclosure include a method ofselecting a treatment regimen for a subject in need thereof, the methodcomprising: receiving a genetic profile for the subject, the geneticprofile comprising a polymorphism in a gene that encodes a cytochromeP450 enzyme; and selecting the treatment regimen based on the geneticprofile. In some embodiments, the treatment regimen comprisesadministering an effective amount of a therapeutic agent. In someembodiments, the treatment regimen comprises withholding a therapeuticagent.

In embodiments, the therapeutic agent is an AXL kinase inhibitor. Insome embodiments of the foregoing, the therapeutic agent is the compoundof structure (I):

In related embodiments, the therapeutic agent is an acid addition saltof the following structure (I).

In more specific embodiments, the salt is a tartrate salt.

In embodiments, therapeutic agent is a compound of structure (IV), or aprodrug or pharmaceutically acceptable salt thereof, as described above.

Embodiments of the present disclosure further include a method fortreating a cancer in a subject, the method comprising: administering aneffective amount of a therapeutic agent to the subject having apredetermined genetic profile comprising a polymorphism in a gene thatencodes a cytochrome P450 enzyme. In embodiments, the polymorphismresults in:

i. an expression level of the cytochrome P450 enzyme that is differentthan a baseline expression level by at least 10%; and/or

ii. an activity of the cytochrome P450 enzyme that is different than abaseline activity by at least 10%.

In embodiments, the expression level of the cytochrome P450 enzyme is atleast 10% greater than the baseline expression level. In embodiments,the activity of the cytochrome P450 enzyme is at least 10% greater thanthe baseline activity. In embodiments, the expression level of thecytochrome P450 enzyme is at least 10% less than the baseline expressionlevel. In embodiments, the activity of the cytochrome P450 enzyme is atleast 10% less than the baseline activity. In embodiments, thetherapeutic agent is an AXL kinase inhibitor.

In certain embodiments, the therapeutic agent is the compound ofstructure (I) or a pharmaceutically acceptable salt thereof. Inembodiments, the therapeutic agent is a pharmaceutically acceptable saleof the compound of structure (I). In embodiments, the pharmaceuticallyacceptable salt of the compound of structure (I) is a tartrate salt. Inembodiments, the therapeutic agent is a compound structure (IV), or aprodrug or pharmaceutically acceptable salt thereof, or a compositioncomprising such a compound, as described above.

In embodiments, a treatment regimen withholding a second therapeuticagent from the subject based on the predetermined genetic profile. Insome embodiments, the second therapeutic agent is the compound ofstructure (I) or a pharmaceutically acceptable salt thereof. Inembodiments, the pharmaceutically acceptable salt of the compound ofstructure (I) is a tartrate salt. In embodiments, the second therapeuticagent is a compound structure (IV), or a prodrug or pharmaceuticallyacceptable salt thereof, or a composition comprising such a compound, asdescribed above.

In some embodiments, the expression level of one or more cytochrome P450enzymes is at least about 10% greater than the baseline expressionlevel. In some embodiments, the expression level is at least about 1%greater, at least about 2% greater, at least about 3% greater, at leastabout 4% greater, at least about 5% greater, at least about 7% greater,at least about 12% greater, at least about 15% greater, at least about17% greater, at least about 20% greater, at least about 22% greater, atleast about 25% greater, at least about 27% greater, at least about 30%greater, at least about 32% greater, at least about 35% greater, atleast about 37% greater, at least about 40% greater, at least about 45%greater, at least about 50% greater, at least about 75% greater, or atleast about 90% greater.

In some embodiments, the activity of one or more cytochrome P450 enzymesis at least about 10% greater than the baseline concentration. In someembodiments, the activity is at least about 1% greater, at least about2% greater, at least about 3% greater, at least about 4% greater, atleast about 5% greater, at least about 7% greater, at least about 12%greater, at least about 15% greater, at least about 17% greater, atleast about 20% greater, at least about 22% greater, at least about 25%greater, at least about 27% greater, at least about 30% greater, atleast about 32% greater, at least about 35% greater, at least about 37%greater, at least about 40% greater, at least about 45% greater, atleast about 50% greater, at least about 75% greater, or at least about90% greater.

A baseline level can be derived from a population. A “population” is anygrouping of subjects or samples of like specified characteristics. Thegrouping could be according to, for example, clinical parameters,clinical assessments, therapeutic regimens, disease status, severity ofcondition, etc.

In some embodiments, the population is selected randomly. In someembodiments, the population is a group comprising about 2, about 5,about 10, about 25, about 50, about 75, or about 100 subjects. In someembodiments, the population is a group comprising about 200, about 300,about 500, about 1,000, about 1,500, about 2,000, about 3,000, about5,000, or about 10,000 subjects. In some embodiments, the population isa group comprising less than about 10,000 subjects. In otherembodiments, the population is a group comprising greater than about10,000 subjects.

In some embodiments, the population is a group that does not havecancer. In embodiments, the population does not have a hematologiccancer. In some embodiments, the population does not have a solid tumorcancer. In some embodiments, the population does not have liver cancer.

In some embodiments, the population is a group that has cancer. Inembodiments, the population has a hematologic cancer. In someembodiments, the population does have a solid tumor cancer. In someembodiments, the population has liver cancer. In some embodiments, thepopulation has a same type of cancer.

In some of the foregoing embodiments, the population is non-responsiveto an AXL kinase inhibitor. In some embodiments, the population isrefractory after treatment with an AXL kinase inhibitor. In someembodiments, the population is intolerant of treatment with an AXLkinase inhibitor. In some embodiments, the AXL kinase inhibitor is thecompound of structure (I) or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt). In some embodiments, the AXL kinase inhibitoris a compound of structure (IV) or a pharmaceutically acceptable saltthereof.

Other embodiments provide a method of determining a metabolic profile ofa subject, the method comprising contacting a population of cells (e.g.,hepatocytes) of the subject with a therapeutic agent and determining aconcentration of a first metabolite which is a compound of any one ofthe foregoing embodiments or a pharmaceutically acceptable salt of anyone of the foregoing embodiments.

In more specific embodiments, the method further comprises determining aconcentration of a second metabolite. In some embodiments, the methodfurther comprises determining an expression level of one or morecytochrome P450 enzymes in the population of cells (e.g., hepatocytes).In other embodiments, the method further comprises determining anactivity of one or more cytochrome P450 enzymes in the population ofcells (e.g., hepatocytes).

In some of the foregoing embodiments, determining the expression levelor determining the activity of the one or more cytochrome P450 enzymesbased on the concentration of the first metabolite.

In other related embodiments, the method further comprises incubatingthe population of cells (e.g., hepatocytes) with the therapeutic agent.In some embodiments, the incubating ranges from about 1.5 to about 2.5hours. For example, in some embodiments the incubating is for about 2hours. In some specific embodiments, the incubating ranges from about0.5 to about 20 hours, about 0.25 to about 10 hours, about 0.15 to about5 hours, about 0.5 to about 10 hours, about 0.5 to about 5 hours, about0.5 to about 3 hours, about 0.25 to about 20 hours, about 0.25 to about5 hours, about 0.25 to about 3 hours, about 0.15 to about 10 hours, orabout 0.15 to about 3 hours.

In some embodiments, the population of cells comprises hepatocytes, forexample, human hepatocytes.

In some embodiments, determining the concentration of the firstmetabolite comprises performing a mass spectrometry assay (e.g., MS,MS/MS, LC-MS, LC-MS/MS). In more specific embodiments, the massspectrometry assay comprises collisionally induced dissociation (CID).In some related embodiments, the determining the concentration of thefirst metabolite further comprises performing a liquid chromatographyassay (LC).

The examples and preparations provided below further illustrate andexemplify compounds of structure (I) or pharmaceutically acceptablesalts of a compound of structure (I), and methods of preparing the salt.It is to be understood that the scope of the present invention is notlimited in any way by the scope of the following examples andpreparations. In the following examples, and throughout thespecification and claims, molecules with a single stereocenter, unlessotherwise noted, exist as a racemic mixture. Those molecules with two ormore stereocenters, unless otherwise noted, exist as a racemic mixtureof diastereomers. Single enantiomers/diastereomers may be obtained bymethods known to those skilled in the art.

G. Examples

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

The examples and preparations provided below further illustrate andexemplify compounds of structure (I) or pharmaceutically acceptablesalts of a compound of structure (I) (e.g., a tartrate salt of thecompound of structure (I)), and methods of preparing such a salt. It isto be understood that the scope of the present invention is not limitedin any way by the scope of the following examples and preparations. Inthe following examples, and throughout the specification and claims,molecules with a single stereocenter, unless otherwise noted, exist as aracemic mixture. Those molecules with two or more stereocenters, unlessotherwise noted, exist as a racemic mixture of diastereomers. Singleenantiomers/diastereomers may be obtained by methods known to thoseskilled in the art.

Example 1: Salt Screening

1 mL of solvent was added to 25 μg of the compound of structure (I) and1 equivalent of the desired acid. The solvents used were water andN-Methyl-2-pyrrolidone (NMP). The salt screen was performed using theCrystal16 equipment. The program allows heating up to 60° C. followed bycontrolled cooling to 5° C. with 0.1° C. per minute. Two series ofslurry experiments were initiated using water and dichloromethane as asolvent. These experiments were stirred for 3 days at 20° C. Ifapplicable, the solvent were evaporated in a vacuum oven to isolate thesolids for XRPD and further analysis as provided below.

X-Ray Powder Diffraction (XRPD)

The X-ray powder diffraction studies were performed using a Bruker AXSD2 PHASER in Bragg-Brentano configuration, equipment #1549. Using a Cuanode at 30 kV, 10 mA; sample stage standard rotating;monochromatisation by a Kβ-filter (0.5% Ni). Slits: fixed divergenceslits 1.0 mm (=0.61°), primary axial Soller slit 2.5°, secondary axialSoller slit 2.5°. Detector: Linear detector LYNXEYE with receiving slit5° detector opening. The standard sample holder (0.1 mm cavity in (510)silicon wafer) has a minimal contribution to the background signal.

Measurement conditions: scan range 5-45° 29, sample rotation 5 rpm,0.5s/step, 0.010°/step, 3.0 mm detector slit; and all measuringconditions are logged in the instrument control file. As systemsuitability, corundum sample A26-B26-S(NIST standard) is measured daily.

The software used for data collection is Diffrac.Commander v3.3.35. Dataanalysis is done using Diffrac.Eva V3.0. No background correction orsmoothing is applied to the patterns. The contribution of the Cu-Kα2 isstripped off using the Diffrac.Eva software.

Thermo Gravitational Analysis/Differential Scanning Calorimetry(TGA/DSC)

The TGA/DSC studies were performed using a Mettler Toledo TGA/DSC1 STAReSystem with a 34-position auto sampler, equipment #1547.

The samples were made using aluminum crucibles (40 μl; pierced).Typically, 5-10 mg of sample was loaded into a pre-weighed aluminumcrucible and was kept at 30° C. for 5 minutes, after which it was heatedat 10° C./min from 30° C. to 300° C. A nitrogen purge of 40 ml/min wasmaintained over the sample. As system suitability check Indium and Zincare used as references.

The software used for data collection and evaluation is STARe Softwarev10.00 build 2480. No corrections are applied to the thermogram.

Differential Scanning Calorimetry (DSC)

The DSC studies were performed using a Mettler Toledo DSC1 STARe System,equipment #1564.

The samples were made using aluminum crucibles (40 μl; pierced).Typically 1-8 mg of sample was loaded onto a pre-weighed aluminumcrucible and was kept at 30° C. for 5 minutes, after which it was heatedat 10° C./min from 30° C. to 350° C. and kept at 350° C. again. Anitrogen purge of 40 ml/min was maintained over the sample. As systemsuitability check Indium and Zinc are used as references.

The software used for data collection and evaluation is STARe Softwarev10.00 build 2480. No corrections are applied to the thermogram.

Microscopy

The microscopy studies were performed using an AxioVert 35M, equippedwith an AxioCamERc 5s, equipment #1612. The microscope is equipped withfour lenses, being Zeiss A-Plan 5×/0.12, Zeiss A-Plan 10×/0.25, LDA-Plan 20×/0.30 and Achros TIGMAT 32×/0.40. Data collection andevaluation is performed using Carl Zeiss Zen AxioVision Blue EditionLite 2011 v1.0.0.0 software.

A small amount of sample is loaded on an object glass and spread until athin layer is obtained.

Dynamic Vapour Sorption (DVS)

The Dynamic Vapour Sorption studies were performed using a SurfaceMeasurement Systems Ltd. DVS-1 No Video, equipment #2126. The sample isloaded into balance pan, typically 20-30 mg, and equilibrated at 0% RH.After the material has dried the RH is increased with 10% per step for 1hour per increment, ending at 95% RH. After completion of the sorptioncycle, the sample was dried using the same method. The software used fordata collection is DVSWin v3.01 No Video. Data analysis is performedusing DVS Standard Analysis Suite v6.3.0 (Standard).

From these experiments in total 16 unique XRPD patterns, or forms, wereobtained. From the salt screen with water as a solvent and controlledcooling with the Crystal16, 11 unique forms were obtained including saltforms from phosphoric acid, tartaric acid (i.e., (+)-L-tartaric acid),fumaric acid, malic acid (i.e., (−)-L-malic acid), succinic acid,ethane-1,5-disulfonic acid, p-toluenesulfonic acid, methanesulfonicacid, benzenesulfonic acid, ethanesulfonic acid and benzoic acid,respectively. Additional acids were tested yielding the correspondingsalt of the compound of structure (I). These additional acids includedhydrochloric acid, sulfuric acid, L-aspartic acid, maleic acid, glutamicacid, citric acid, D-glucuronic acid, glycolic acid, D-gluconic acid,L-ascorbic acid, adipic acid, naphthalene-1,5-disulfonic acid andnaphthalene-2-sulfonic acid.

A salt screen with NMP as a solvent and controlled cooling with theCrystal16 did lead to one unique form from sulfuric acid, at low yieldand not enough material could be obtained for further analysis.

The slurry experiments with dichloromethane as a solvent lead to 4 newforms from maleic acid, malic acid, succinic acid, andethane-1,5-disulfonic acid, respectively. The solids from the saltscreens were isolated for XRPD analysis, each unique new pattern wasanalyzed using DSC-TGA, microscopy, FT-IR, ¹H NMR and HPLC. In thisstudies, some salt forms were found to be non-reproducible and othersshowed reduced crystallinity after DVS.

Five forms formed from phosphoric acid, tartaric acid, malic acid,succinic acid, and benzenesulfonic acid were selected for scale upexperiments, up to 500 mg, for further analysis.

Upon screening of numerous salt forms of the compound of structure (I),tartrate salt of the compound of structure (I) was identified as asuitable salt form of the compound for pharmaceutical uses.Surprisingly, PK studies showed improved bioavailability of the tartratesalt and the PhysChem properties measured showed the tartrate as havingdesirable physical properties including good stability. See Examplesbelow.

Example 2: Pharmacokinetic Testing of Salt Forms

The 5 salt forms of structure (I) described in Example 1 (i.e., formedfrom phosphoric acid, tartaric acid, malic acid, succinic acid andbenzenesulfonic acid) were tested to determine their pharmacokinetic(PK) profiles. Fasted male Sprague-Dawley rats were dosed with an oralformulation of each salt form as well as the free base form. Plasmaconcentration was tested at 5 minutes, 0.25, 0.5 1, 2, 4, 8, 12 and 24hours post-dose. The mean plasma concentration after PO dosing isillustrated in FIG. 1. The data (mean values) for the 5 different saltforms and the free base is included in Table 8, below.

TABLE 8 PK data comparison for 5 representative salt forms of thecompound of structure (I). Benzene Compound of Phosphate Tartrate MalateSuccinate sulfonate Structure (I) Salt Salt Salt Salt Salt Dose (PO,mg/kg) 18.2 14.3 18.7 18.6 18.7 17.6 C_(max) (ng/mL) 116 103 174 13479.9 117 T_(max) (hours) 0.833 1.00 0.667 0.500 0.833 0.667AUC_(0-24 hours) 458 531 581 515 470 408 (ng · h/mL) Bioavailability (%)26.9 47.2 42.9 37.3 32.9 32.4

As the data in Table 8 show, the tartrate salt unexpectedly had one ofthe best overall PK profiles, having the highest C_(max), highest AUCfor 0-24 hours, and second highest bioavailability. Because the saltobtained from phosphoric acid showed undesirable stabilitycharacteristics, it appears that the tartrate salt has the best overallprofile as a drug substance.

Example 3: Pharmacokinetic Study—Free Base v. Tartrate Salt

Fasted male Sprague-Dawley rats were dosed with the free base and thetartrate salt form of structure (I) in 20% solutol. The free base wasformulated at 5.0 mg/mL (PO) and dosed by oral gavage (18.2 mg/kg). Thetartrate salt of structure (I) was formulated at 6.5 mg/mL to accountfor the added weight of the tartrate component of the salt and dosed byoral gavage (14.5 mg/kg).

Plasma samples were taken at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hourspost dose and analyzed for the concentration of structure (I) byLC-MS/MS with reference to a previously determined standard curve.Pharmacokinetic parameters were calculated using a noncompartmentalapproach with Phoenix WinNonlin 6.3 (Pharsight, Mountain View Calif.).

It was observed that the tartrate salt was superior to the free basehaving a higher peak of bioavailability than the free base formulation(FIG. 2). These data suggest that the tartrate salt of structure (I) maybe more useful in vivo than the free base form. Additionally, thetartrate salt form shows superior C_(max) and AUC parameters (FIG. 2)while maintaining an equivalent toxicity profile to the free base format equal doses. That is, the tartrate salt of structure (I) allows forhigher drug plasma levels without additional toxicity. Pharmacokineticdata for the tartrate salt v. the free base is included in Table 9,below (nominal dose of 20 mg/kg).

TABLE 9 PK data comparing the free base of the compound of structure (I)to the tartrate salt. Free Base Tartrate Salt Dose (PO, ^(mg)/_(kg))18.2 18.7 C_(max) (^(ng)/_(mL)) 116 174 T_(max) (hours) 0.833 0.667AUC_(0-24 hours) (^(ng · h)/_(mL)) 458 581 Bioavailability (%) 26.9 42.9

Example 4: Synthesis of a Tartrate Salt of Structure (I)

2-nitrobenzenesulfonyl chloride was combined with triethylamine anddimethylamine in acetonitrile under the reaction conditions shown toafford N,N-dimethyl-2-nitrobenzenesulfonamide in 82% yield.

N,N-dimethyl-2-nitrobenzenesulfonamide was combined with zinc andammonium chloride in methanol under the reaction conditions shown toafford 2-amino-N,N-dimethylbenzenesulfonamide in 99% yield.

N,N-dimethylbenzenesulfonamide was combined with2,4,5-trichloropyrimidine and tetrabutylammoniumhydrogen sulfate underthe reaction conditions shown to afford2-((2,5-dichloropyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide in32% yield. ¹HNMR characterization data is shown in FIG. 103A.

2-((2,5-dichloropyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide wascombined with 4-aminobenzoic acid and tetrabutylammoniumhydrogen sulfateunder the reaction conditions shown to afford4-((5-chloro-4-((2-(N,N-dimethylsulfamoyl)phenyl)amino)pyrimidin-2-yl)amino)benzoicacid in 85% yield. ¹HNMR characterization data is shown in FIG. 103B.

4-((5-chloro-4-((2-(N,N-dimethylsulfamoyl)phenyl)amino)pyrimidin-2-yl)amino)benzoicacid was combined with borane in tetrahydrofuran (1M) under the reactionconditions shown followed by treatment with 4 M hydrochloric acid underthe reaction conditions shown to afford2-((5-chloro-2-((4-(hydroxymethyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidein 80% yield. ¹HNMR characterization data is shown in FIG. 103C.

2-((5-chloro-2-((4-(hydroxymethyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidewas combined with thionyl chloride in dichloromethane under the reactionconditions shown to afford2-((5-chloro-2-((4-(chloromethyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidein 90% yield. ¹HNMR characterization data is shown in FIG. 103D.

2-((5-chloro-2-((4-(chloromethyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidewas combined with potassium carbonate and 1-methylpiperazine inacetonitrile under the reaction conditions shown to afford2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide(i.e., structure (I) or Compound 1) in 80% yield.

2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide(i.e., structure (I) or Compound 1) was treated with tartaric acid toafford2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidemono-tartrate salt.

Example 5: Determination of Dosage and Treatment Outcomes

Groups of pluralities of patients (e.g., 3) are treated with a compoundof structure (I), or a pharmaceutically acceptable salt thereof (e.g., atartrate salt) using escalated doses until a maximum tolerated dose(MTD) is established (“the MTD expansion safety cohort”). Typically, inthe absence of dose limiting toxicities (DLTs) doses are increased usinga modified Fibonacci dose escalation scheme.

When the MTD is established, dosages are adjusted to an average doseadministered in the MTD expansion safety cohort. Additional cohorts(e.g., 5) of patients with specific tumor types may be treated with acompound of structure (I), or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt) to confirm safety, explore biomarkers, andevaluate potential signals of activity of a compound of structure (I),or a pharmaceutically acceptable salt thereof (e.g., a tartrate salt) isadministered to specific groups of heavily pretreated patients (e.g.,tumor patients that have progressed despite immunotherapy, EGFR+ NSCLCpatients that have progressed on ≤2 lines of tyrosine kinase inhibitors,BRAF-, KRAS- or NRAS-mutated colorectal carcinoma patients for whom nostandard therapy remains, persistent/recurrent ovarian cancer patientsthat are or would be platinum refractory/resistant and BRAF-mutatedmelanoma patients that have not responded to immunotherapy or acombination of BRAF/MEK inhibitor) or given in combination withimmunotherapy or a tyrosine kinase inhibitor.

In particular, when the MTD is confirmed, a single daily dose of acompound of structure (I), or a pharmaceutically acceptable salt thereof(e.g., a tartrate salt), is administered orally on days 1-21 of a 28 daycycle based on the average of the dose administered in the MTD expansionsafety cohort.

Treatment outcomes may be evaluated by dose limiting toxicities andtreatment emergent adverse events. Dose limiting toxicities andtreatment emergent adverse events may include Grade 3 or greater febrileneutropenia, Grade 4 ANC for 7 or more consecutive days, Grade 4thrombocytopenia or Grade 3 thrombocytopenia with clinically significantbleeding or that requires a platelet transfusion, Grade 3 or 4non-hematologic adverse events including nausea, vomiting, diarrhea, andelectrolyte imbalances persisting for more than 48 hours despite optimalmedical management.

Treatment outcomes can also be evaluated by measuring plasmaconcentrations from blood taken from subjects at pre-dose, day 1 and day21 (derived PK parameters by non-compartment analysis). For example,blood can be collected at 0.5, 1 2, 4, 8, 24 hours post-dose on day 1and at 48 hours post dose on day 21. Plasma concentration calculatedover time can be used to determine an area under the curve for time 0 toinfinity, from time 0 to the last measured time point, and peak plasmaconcentration. Biomarkers in tumor tissue, PBMCs, plasma and serum arealso assessed (e.g., using Spearman rank correlation statistic). PBMCsand serum are obtained prior to the first dose on day 1 and at 2, 6, and24 hours after dosing, and again on day 8. A baseline tumor assessmentis also performed and repeated after the 28 day cycle (i.e., cycle 2 andon even cycles thereafter).

Example 6: Combination of CDK Inhibitor and AXL Kinase Inhibitor forTreatment of Cancer

DOHH2 cells (a B-cell lymphoma cell line) were treated with eitheralvocidib alone or in combination with a tartare salt of the compound ofstructure (I) at 1 or 3 nM fixed concentrations and alvocidib atconcentrations ranging from 1-1000 nM for 72 hours, as shown in FIG. 3.Single dose concentrations of a tartrate salt of the compound ofstructure (I) were added to varying alvocidib dilutions. Viability wasassessed using CellTiter-Glo according to manufacturer protocol.

HCT-116 cells (a KRAS mutant colorectal cancer cell line) were treatedwith either alvocidib alone or a combination of a tartrate salt of thecompound of structure (I) at 1 or 3 nM fixed concentrations andalvocidib at concentrations ranging from 0.01-100 nM for 72 hours asshown in FIG. 4 for 72 hours. Single dose concentrations of a tartratesalt of the compound of structure (I) were added to varying alvocidibdilutions. Viability was assessed using CellTiter-Glo according tomanufacturer protocol.

The data in FIGS. 3 and 4 show that a CDK inhibitor, such as alvocidib,synergizes with an AXL kinase inhibitor, such as a tartrate salt of thecompound of structure (I), to potently reduce cell viability of cancercells.

Example 7: Increased Active DCs in Tumors Following Administration of anAXL Kinase Inhibitor

Mice were treated with a tartrate salt of the compound of structure (I)at a concentration of 60 mg/kg. Tumors were assayed by flow cytometry tomeasure the presence of active DCs using a CD86 antibody and a CD11cantibody. FIG. 5 shows that the percent of activated DCs in tumors cellsis increased following treatment with an AXL kinase inhibitor ascompared to treatment with a negative control (vehicle).

Example 8: Combination of AXL Kinase Inhibitor and a PD1/PD-L1Checkpoint Inhibitor for Treatment of Cancer

Mice were treated with a tartrate salt of the compound of structure (I)alone (at 60 mg/kg), a PD-L1 antibody alone (200 μg), or both incombination. Tumor volume was measured for fifteen days followinginoculation.

FIGS. 6A and 6B show treatment with a tartrate salt of a compound ofstructure (I) effects in the 4T1 breast cancer syngraft in mice. (FIG.6A) Tumor volumes are shown following the treatment of a tartrate saltof a compound of structure (I) at a concentration of 60 mg/kg and/oranti-PD-L1 antibody (200 μg/mouse/dose). Combination treatment appearsto have the greatest anti-tumor effect in this model. (FIG. 6B)Comparable body weight losses were observed for single agent using atartrate salt of a compound of structure (I), and the combinationtreatment with anti-PD-L1 antibody.

The tartrate salt of the compound of structure (I) achieved a tumorgrowth inhibition (% TGI) of 67.1% as a single agent, while anti-PD-L1achieved % TGI of 41.5% in this model. The combination induced a % TGIof 87.3%, over the 16 day treatment schedule.

% TGI is the tumor volume for a given treatment group compared to thevehicle control group, i.e., (1-(TVtreatment/TVvehicle))*100. Tumorvolumes were measured in two dimensions using a caliper, and the volumewas expressed in mm3 using the formula: V=(L×W×W)/2, where V is tumorvolume, L is tumor length (the longest tumor dimension) and W is tumorwidth (the longest tumor dimension perpendicular to L).

In a second set of experiments, mice were treated with a tartrate saltof the compound of structure (I) alone (at 25 mg/kg as the “low dose”and 40 mg/kg as the “high” dose), an anti-PD-1 antibody alone (10mg/kg), or both in combination, with or without single dose radiationtherapy at 12 GY/animal. Tumor volume was measured for several days.FIG. 7 shows the efficacy of the combination treatment in a mouseallograft model of breast cancer (4T1). FIG. 8 shows the efficacy of thecombination treatment in a mouse model of melanoma (B16). FIG. 9 showsthe efficacy of the combination treatment in a mouse model of colorectalcancer (CT26). FIG. 10 shows the efficacy of the combination treatmentin a mouse model of lung cancer (Lewis lung).

In a third experiment, the in vivo activity of a tartrate salt of thecompound of structure (I) as a single agent, and combinations withimmune checkpoint blockade (anti-PD-1) and radiation therapy (RT), wereassessed in the 4T1 syngeneic (allograft) mouse model for breast cancer.A tartrate salt of the compound of structure (I) was dosed qd by oralgavage at 25 mg/kg. Anti-PD-1 antibody was dosed at 10 mg/kg, biw,intraperitoneally. Mice dosed with radiation were given a single dose of12 GY. FIG. 11A shows tumor volumes and FIG. 11B shows body weights foranimals on study.

A tartrate salt of the compound of structure (I) achieved a tumor growthinhibition (% TGI) of 28.4% as a single agent, while anti-PD-1 and RTeach alone achieved % TGI of 10.2% and 43.7%, respectively. However, thecombination of all three induced a % TGI of 57.7%, over the 18 daytreatment schedule.

The pharmacokinetic profile of a tartrate salt of the compound ofstructure (I) in a 4T1 model was also assessed. FIG. 114 showspharmacokinetic profile of a tartrate salt of the compound of structure(I) in 4T1 model. 4T1 bearing mice were treated with 60 mpk of atartrate salt of the compound of structure (I) tartrate p.o. Tumor andblood were collected at the indicated time points.

The effect of a tartrate salt of the compound of structure (I) oncytokines in serum in a 4T1 model was also assessed (FIG. 115). Balb/cmice were transplanted with 4T1 cells orthotopically. 7 days aftertransplantation, the compound of structure (I) tartrate wasadministrated (60 mg/kg, p.o., Q.D.). Whole blood was collected 2, 6,and 24 hour after the last dosage on Day 12. Cytokines in serum weremeasured with Milliplex assay. Normal indicates healthy mouse withouttumor, n=6 (vehicle: n=5, normal: n=3). Error Bar indicates SD. N.Dindicates “no data”.

Example 9: Combination of AXL Kinase Inhibitor and an EGFR Inhibitor forthe Treatment of Cancer

Using the H1650 xenograft model, which is an EGFR mutated lung cancermodel, mice were treated with a tartrate salt of the compound ofstructure (I) alone (at 12.5 or 25 mg/kg), an EGFR inhibitor (erlotinib)alone (75 mg/kg), or both in combination. Tumor volume was measured forseveral days. FIG. 12 shows that a tartrate salt of the compound ofstructure (I) and the EGFR inhibitor have synergistic effects in an EGFRmutated lung cancer model.

Using the H1650 xenograft model, mice were treated with a tartarate saltof the compound of structure (I) alone (40 mg/kg), an EGFR inhibitoralone (either erlotinib-HCl at 20 mg/kg or osimertinib at 10, 20, or 40mg/kg), or combinations of the tartarate salt of the compound ofstructure (I) and the EGFR inhibitors. Tumor volume was measured forseveral days. FIG. 13 shows the efficacy of the combinations of thetartarate salt of the compound of structure (I) and the EGFR inhibitors.

Example 10: AXL Inhibition Leads to a Reversal of a MesenchymalPhenotype, Sensitizing Cancer Cells to Targeted Agents andImmuno-Oncology Therapies

Mesenchymal properties and the epithelial-to-mesenchymal transition(EMT) contribute to the initiation and progression of many tumor typesand ultimately can lead to drug resistance and highly aggressivedisease. It was found that a tartrate salt of a compound of structure(I), a potent AXL inhibitor, leads to a reversal of the mesenchymalphenotype in multiple cancer models. Following treatment with a tartratesalt of a compound of structure (I), changes in mRNA expression wereobserved using RT-qPCR and protein expression using standardimmunoblotting that are consistent with a reversal of the mesenchymalphenotype (see, for example, FIGS. 14 and 15).

FIG. 14 shows protein levels of pAXL and total AXL. Panc-1 cells werepre-treated with 0.1-1 μM of a tartrate salt of a compound of structure(I) for 1 hour, then AXL phosphorylation was induced by a 10-minutetreatment of an apoptotic cell lysate and GAS6. Lysate and GAS6treatment leads to phosphorylation of AXL. A tartrate salt of a compoundof structure (I) inhibits the phosphorylation of AXL in a dose-dependentfashion.

FIG. 15A and FIG. 15B show changes in EMT marker expression after 2hours of treatment with a tartrate salt of a compound of structure (I).Cells were treated with a single dose of a tartrate salt of a compoundof structure (I). Snail (SNAI1) and Slug (SNAI2) levels were measuredvia RT-qPCR after 2 hours. A concentration-dependent inhibition ofexpression levels is observed in (FIG. 15A) MV4-11 and (FIG. 15B) A549cells.

Upon treatment with a tartrate salt of a compound of structure (I),cancer cells possessed lower motility and a decrease inanchorage-independent growth, both hallmarks of a mesenchymal cell (seefor example, FIG. 16A and FIG. 16B).

FIG. 16A and FIG. 16B show effects of treatment with a tartrate salt ofa compound of structure (I) on the migration of Pane-1 or Aspc-1 cells.(FIG. 16A) The effect of treatment with a tartrate salt of a compound ofstructure (I) on the migration of Panc-1 cells was assessed in a scratchassay. Confluent cells were scratched, and then treated for 24 hourswith GAS6, 0.5 μM of a tartrate salt of a compound of structure (I), orR428. Treatment with a tartrate salt of a compound of structure (I)reduces migration following treatment. (FIG. 16B) The effect of atartrate salt of a compound of structure (I) treatment on the anchorageindependent migratory capability of Aspc-1 cells in a soft agar assay.Treatment with a compound of structure (I) again reduces migration inthis assay.

In vivo models of erlotinib-resistant non-small cell lung cancer (NSCLC)were utilized to demonstrate single agent activity of a tartrate salt ofa compound of structure (I) in highly mesenchymal models. Additionally,treatment with a tartrate salt of a compound of structure (I) was ableto sensitize this highly refractory model to erlotinib (see for exampleFIGS. 17 and 18).

FIG. 17A and FIG. 17B show treatment effects of a tartrate salt of acompound of structure (I), in combination with erlotinib, in an in vivoxenograft model for lung cancer. A549 cells were injected subcutaneouslyinto the hind flank of athymic nude mice at 1×10⁷ cells/mouse. Oncetumor volumes reached 100 mm³, mice were randomized into study arms.Mice were treated with a “two day on, two day off” dosing schedule witha tartrate salt of a compound of structure (I) (75 mg/kg) and/orerlotinib (25 mg/kg). This dosing level and schedule was tolerated wellin the animals (FIG. 17B). Combination treatment resulted in maintenanceof tumor volumes (FIG. 17A), and was synergistic relative to treatmentof either drug as a single agent.

FIGS. 18A and 18B show treatment effects of a tartrate salt of acompound of structure (I), in combination with erlotinib, in an in vivoxenograft model for lung cancer. H1650 cells were injectedsubcutaneously into the hind flank of athymic nude mice at 1×10⁷cells/mouse. Once tumor volumes exceeded 100 mm³, mice were randomizedinto study arms. Mice were treated with a “three day on, three day off”dosing schedule with a tartrate salt of a compound of structure (I) at aconcentration of 12.5 or 25 mg/kg, and/or erlotinib (75 mg/kg). Thisdosing level and schedule was tolerated well in the animals (FIG. 18B).Combination treatment resulted in significant regression of tumorvolumes (FIG. 18A), and was synergistic relative to treatment of eitherdrug as a single agent.

Inhibition of AXL by can inhibit tumor-associated efferocytosis leadingto a stronger immunogenic response to the tumor. As shown in FIG. 19Aand FIG. 19B, treatment with a tartrate salt of a compound of structure(I) impairs the AXL-mediated phagocytosis of apoptotic bodies.Efferocytosis assays interrogating the effects of a tartrate salt of acompound of structure (I) treatment in phorbol ester (PMA) treated THP-1cells (macrophage induction) were performed. (FIG. 19A) THP-1 cells werepre-treated with a tartrate salt of a compound of structure (I) andGAS6, followed by treatment with fluorescently labeled apoptotic lysate(staurosporine-treated A549 cell lysate). Following 24-hour incubation,flow cytometry was performed to assess the ability of THP-1 cells tophagocytose apoptotic bodies. (FIG. 19B) A similar experiment isperformed using fluorescence microscopy in THP-1 cells. Fluorescentlylabeled apoptotic bodies were counted in adherent THP-1 cells usingImageJ software on confocal microscopy images (Olympus FV1000).

Furthermore, a tartrate salt of a compound of structure (I) demonstratedsynergy when combined with an anti-PD-L1 agent, which is an immunecheckpoint inhibitor, in a syngeneic triple negative breast cancer mousemodel (see for example, Example 8 and FIGS. 6A and 6B).

Interestingly, during the evaluation of a tartrate salt of a compound ofstructure (I) in models of EMT, a dramatic change was detected in theexpression of the retinoic acid (RA) metabolizing protein CYP26A1 (seefor example FIGS. 20-22), suggesting that AXL inhibition leads tochanges in RA metabolism.

FIG. 20 shows CYP26A1 mRNA levels in RA and cell treated with a tartratesalt of a compound of structure (I). Cells were treated with 1 μMRetinyl Acetate (RA) and/or a tartrate salt of a compound of structure(I) at 0.5 μM. Treatment with RA and a tartrate salt of a compound ofstructure (I) induced RA-dependent expression of CYP26A1.

FIGS. 21A and 21B show CYP26A1 mRNA expression in MV4-11 cells. (FIG.21A) Cells were treated with non-targeting or AXL specific siRNA. Cellswere subsequently treated with 1 μM Retinyl Acetate (RA) and a tartratesalt of a compound of structure (I). Treatment with AXL siRNA inducedrobust increase in CYP26A1 expression in all samples, and the strongestincrease in the RA treated sample. (FIG. 21B) Immunoblottingconfirmation of protein knockdown by AXL siRNA.

FIGS. 22A and 22B show AXL co-immunoprecipitates with RA importassociated gene, Stra6. (FIG. 22A) From a small cell panel, it can beseen that AXL is highly expressed in cells such as HCT116 or Panc-1.(FIG. 22B) Using the HCT116 cell line, it can be seen aco-immunoprecipitation of Stra6 and AXL. Stra6 is a primary cellimporter of RA.

These data suggest that AXL induces a transition to a mesenchymalphenotype in cancer cells through the suppression of RA signaling andthat a tartrate salt of a compound of structure (I) can rapidly reversethis phenotype causing the cell to revert to a more differentiatedstate. Thus, a tartrate salt of a compound of structure (I) has singleagent activity and combined synergy with targeted anti-cancer agents andimmunotherapies.

In summary, a compound of structure (I) inhibits theapoptotic-cell/GAS6-mediated induction of AXL phosphorylation; inhibitsexpression of mesenchymal genes, Snail and Slug; inhibits the migrationof pancreatic cancer cells; the combination of a compound of structure(I) and erlotinib is an active regimen in multiple lung cancer xenograftmodels; a compound of structure (I) inhibits AXL-mediated endocytosis ofapoptotic bodies (efferocytosis); a combination of a compound ofstructure (I) and Anti-PD-L1 is an active regimen in the 4T1 breastcancer syngraft model; and a compound of structure (I) increases theCYP26A1 induction mediated by RA.

Example 11: Inhibition of AXL Kinase Suppresses Retinoic Acid Metabolism

Retinoic acid (RA), a metabolite of Vitamin A (retinol), has been usedas a single-agent treatment in patients with acute promyelocyticleukemia (APL) with roughly 90% of patients attaining a completeremission. However, remissions can be transient and resistance ariseswithin a few months following treatment.

This example aims to determine the role of AXL inhibition as a means ofrestoring sensitivity to RA treatment. It was hypothesized thattreatment with the AXL inhibitor, a compound of structure (I), woulddisrupt RA metabolism.

Following treatment with a tartrate salt of a compound of structure (I),changes in mRNA expression were analyzed using RT-PCR, proteinexpression using standard immunoblotting, and endogenous RA levels usinga competitive ELISA (see, for example FIG. 23). FIG. 23 shows proteinlevels of pAXL and total AXL. A549 cells were pre-treated with 0.5 μM ofa tartrate salt of a compound of structure (I) for 1 hour, then AXLphosphorylation was induced by a 30-minute treatment of an apoptoticcell lysate and GAS6. Lysate and GAS6 treatment leads to phosphorylationof AXL. This result demonstrates that a a compound of structure (I)inhibits the phosphorylation of AXL.

RT-PCR was used to measure mRNA expression of CYP26 in cells inducedwith RA and treated with a tartrate salt of a compound of structure (I).Following treatment, changes in CYP26 expression were assessed at theprotein level, using standard western blotting techniques. Endogenous RAlevels were measured using a competitive ELISA technique. To determinethe effect of a compound of a tartrate salt of structure (I) on tumorgrowth in an in vivo model, treatment with a tartrate salt of a compoundof structure (I) was tested in a MV4-11 xenograft mouse model.

A robust induction in mRNA expression levels of CYP26 was observedfollowing 1 μM RA treatment in MV4-11 leukemia cells, reaching nearly4.3-fold after 6 hours of treatment (FIG. 30A). However, treatment ofcells with a tartrate salt of a compound of structure (I) at levels aslow as 100 nM inhibited RA-mediated induction of CYP26 mRNA levels by88.9% in MV4-11 cells at 6 hours (see, for example, FIG. 3).Interestingly, administration of a tartrate salt of a compound ofstructure (I) also inhibited basal mRNA levels of CYP26 by 94.1% at 6hours (see, for example, FIGS. 24 and 25).

Changes in neural crest EMT following treatment with a tartrate salt ofa compound of structure (I) suggest RA-dependent gene expression(CYP26A1). MV4-11 cells treated with a single dose of a tartrate salt ofa compound of structure (I) suggested that a tartrate salt of a compoundof structure (I) inhibits EMT in a RA signaling-dependent fashion.

Similar trends in CYP26A1 expression are observed in additional celllines (HL60, A549, and H1650; see, for example, FIG. 25 and FIG. 30B),and with an alternative AXL inhibitor, R428, although only at higherconcentrations (see, for example, FIGS. 26 and 33).

FIG. 24 shows changes in CYP26A1 expression in (top panel) MV4-11 andHL-60 cells (bottom panel) treated with a single dose of a tartrate saltof a compound of structure (I). CYP26A1 mRNA levels were measured viaRT-qPCR at several time points. An initial suppression of CYP26A1expression is followed by a rebounding increase of CYP26A1 expressionstarting around 24 hours. FIG. 25 shows the same effects observed inA549 cells.

FIG. 26 shows CYP26A1 expression in R428 treated cells. MV4-11 cellswere treated with a single dose of R428. CYP26A1 mRNA levels weremeasured via RT-qPCR at 24 hrs. Induction of CYP26A1 was seen with R428treatment, though the magnitude was not as large as that seen with atartrate salt of a compound of structure (I).

As shown in FIG. 27, changes in CYP26A1 expression and intracellular RAlevels were analyzed. Cells were treated with a single dose of atartrate salt of a compound of structure (I) and Retinyl Acetate (RA),and cells were harvested at 6 hrs. CYP26A1 levels were measured viaRT-qPCR, and endogenous levels of RA were measured by a competitiveELISA(mybiosource.com). Where CYP26 mRNA is suppressed by treatment witha tartrate salt of a compound of structure (I) at a concentration of 0.1μM, RA levels were salvaged.

At time points greater than 24 hours, and without re-treatment with atartrate salt of a compound of structure (I), CYP26 expression exceededlevels observed in induced samples. In cells treated with a tartratesalt of a compound of structure (I), it was observed that RA levels weremaintained at time points where CYP26 expression was suppressed. Atartrate salt of a compound of structure (I) strongly inhibited tumorvolumes by up to 100% at multiple dose levels and treatment schedules(see for example FIGS. 34A-34D).

FIG. 34A and FIG. 34B show MV4-11 xenograft studies; and FIG. 34C andFIG. 34D show A549 xenograft studies. Xenograft studies were performedin athymic nude mice with the doses and schedules of a tartrate salt ofa compound of structure (I) indicated above. Mice were given food andwater ad libitum. Doses shown did not cause significant bodyweightlosses in the treated animals. Significant tumor growth inhibition wasobserved in both tumor models investigated.

Changes in CYP26A1 expression in vivo are shown in FIG. 35. Athymic nudemice bearing MV4-11 tumors xenografted subcutaneously were given asingle dose of 180 mg/kg of a tartrate salt of a compound of structure(I). Tumors were harvested 6 and 24 hrs post treatment. CYP26 proteinexpression in tumors and livers is shown in FFPE tissues by standard IHCstaining, using fast red chromogen (A). mRNA levels of CYP26 are shownfor both tumor (B) and livers (C).

Analysis of CYP26 expression in fixed tissues, and RA levels in plasmawill be assessed to determine the effects of a tartrate salt of acompound of structure (I) on physiological levels of CYP26 and RA.

These observations demonstrate that inhibition of AXL kinase by atartrate salt of a compound of structure (I) suppresses CYP26 anddisrupts RA metabolism. Taken together, these data indicate that AXLlikely serves as a suitable therapeutic target for addressing cellularresponses mediating RA resistance.

In summary, a compound of structure (I) inhibits theapoptotic-cell/GAS6-mediated induction of AXL phosphorylation; acompound of structure (I) inhibits expression of mesenchymal genes,Snail and Slug; a compound of structure (I) phenocopies retinoic acideffects, in an RA-dependent fashion, including inducing expression ofCYP26A1 in multiple cell lines; the AXL inhibitor, R428, also inducesCYP26A1 expression, though to a lesser degree; compound of structure(I)-mediated modulation of CYP26A1 levels corresponds with alteredintracellular RA levels; a compound of structure (I) increases theCYP26A1 induction mediated by RA; AXL siRNA also induces CYP26A1increases; AXL co-precipitates with Stra6, and may serve as a negativeregulator of Stra6; and a compound of structure (I) is an activecompound in multiple xenograft tumor models.

Example 12: Inhibition of AXL Kinase Reverses the Mesenchymal Phenotypein Leukemic Cells

Following treatment with a tartrate salt of a compound of structure (I),changes in mRNA expression were interrogated using RT-PCR, proteinexpression was measured using standard immunoblotting, and endogenous RAlevels were measured using a competitive ELISA (see for example, FIGS.28 and 29).

FIG. 28 shows that a tartrate salt of a compound of structure (I)affects the expression of multiple genes involved in RA synthesis andmetabolism. A RT2 Profiler PCR array (Qiagen) was used to profileexpression changes with RA and/or 0.1 μM treatment with a tartrate saltof a compound of structure (I). Multiple genes are affected, includingCYP26A1, a major CYP involved in RA degradation.

FIG. 29 shows a selected list of genes that respond to RA and a tartratesalt of a compound of structure (I).

Also, the effect of a tartrate salt of a compound of structure (I) ontumor growth was assessed in an in vivo model, assessing efficacy of atartrate salt of a compound of structure (I) in an MV4-11 xenograftmouse model (see for example, FIG. 30).

FIGS. 30A and 30B show CYP26A1 mRNA expression in (FIG. 30A) MV4-11 and(FIG. 30B) A549 cells. Cells were treated for 6 hours with 1 μM RetinylAcetate (RA) and a tartrate salt of a compound of structure (I) at 0.1μM or 0.5 μM. Treatment with RA and a tartrate salt of a compound ofstructure (I) suppressed RA induced expression of CYP26.

One of the genes that was detected as being dramatically changed bytreatment with a tartrate salt of a compound of structure (I) was the RAmetabolizing protein CYP26A1 (see for example, FIGS. 31 and 32),suggesting that AXL inhibition indeed leads to changes in RA metabolism.

FIG. 31 shows CYP26A1 protein expression in A549 cells. Cells weretreated for 24 hours with 1 μM Retinyl Acetate (RA) and a tartrate saltof a compound of structure (I) at 0.1 μM or 0.5 μM. Treatment with RAand a tartrate salt of a compound of structure (I) inhibits expressionof CYP26A1 in a dose dependent fashion.

FIG. 32 shows changes in CYP26A1 expression over 72 hours with low-dosetreatment with a tartrate salt of a compound of structure (I). Cellswere treated with Retinoic Acid (RA) with/without multiple doses of atartrate salt of a compound of structure (I), given at 0, 24, and 48hrs. CYP26A1 levels were measured via RT-PCR. CYP26A1 expression quicklyinduced by RA, but inhibited by a tartrate salt of a compound ofstructure (I) at both 0.1 μM and 0.5 μM. Repeated dosing prevents therebounding expression of CYP26A1 mRNA seen in prior experiments.

A strong induction of CYP26 mRNA expression following RA treatment inMV4-11 leukemia cells was observed, and this effect was also observed intreatment with an AXL inhibitor, a tartrate salt of a compound ofstructure (I), at levels as low as 100 nM. Activity of a tartrate saltof a compound of structure (I) was also assessed in additional celllines (HL60, A549, and H1650, see, for example, FIG. 24), and with analternative AXL inhibitor, R428 (see, for example, FIG. 33). FIG. 33shows changes in CYP26A1 expression over 24 hours for MV4-11 cellstreated with a single dose of R428. CYP26A1 mRNA levels were measuredvia RTPCR at 24 hrs. An initial suppression of CYP26A1 expression isfollowed by a rebounding increase of CYP26A1 expression starting around24 hours. Suppression seen with R428 was not as large as with a tartratesalt of a compound of structure (I).

Treatment with a tartrate salt of a compound of structure (I) correlatedwith increased CYP26 expression and reduced levels of endogenous RA. Invivo, a tartrate salt of a compound of structure (I) strongly inhibitedxenograft tumor volumes by up to 100% with multiple dose levels andtreatment schedules (see, for example, FIGS. 34A-34D). CYP26 expressionin fixed tissues correlated well with mRNA levels observed in xenografttumors following treatment (see, for example, FIGS. 35-37).

FIGS. 35-37 shows changes in CYP26A1 expression in vivo. Athymic nudemice bearing MV4-11 tumors xenografted subcutaneously were given asingle dose of 180 mg/kg of a tartrate salt of a compound of structure(I). Tumors were harvested 6 and 24 hrs post treatment. CYP26 proteinexpression in tumors and livers is shown in FFPE tissues by standard IHCstaining, using fast red chromogen (FIG. 35). mRNA levels of CYP26 areshown for both tumor (FIG. 36) and livers (FIG. 37).

Taken together, these observations demonstrate that inhibition of AXLkinase by a tartrate salt of a compound of structure (I) can disrupt RAmetabolism by inducing CYP26 expression and this disruption of RAmetabolism leads to reversal of the mesenchymal phenotype in leukemiccells.

In summary, a compound of structure (I) alters gene expression ofmultiple genes involved in RA synthesis, degradation, and signaling; acompound of structure (I) potently inhibits retinyl acetate and retinoicacid-induced expression of the RA degrading CYP protein, CYP26,expression in multiple cell lines; a compound of structure (I) cancontinually repress CYP26 expression with repeated dosing; a compound ofstructure (I)-mediated reduction in CYP26 corresponds with higherintracellular RA levels; the AXL-specific inhibitor, R428, inducessimilar effects, though less potently a compound of structure (I) is anactive compound in multiple xenograft tumor models; a compound ofstructure (I) may synergize with retinoid therapies, including ATRA.

Example 13: Predictive and Pharmacodynamic Biomarkers for Cancer Treatedwith an AXL Kinase Inhibitor

As shown in FIG. 38, serum levels of GAS6 were measured at varioustime-points during and following treatment with a tartrate salt of thecompound of structure (I) for study subjects A-G. Serum levels of GAS6were measured at the following times: Cycle 1/Day 1 pre-dose (“C1D1PRE”), and 2 and 24 hours post-dose (“C1D1 2” and “C1D1 24”,respectively) and Cycle 1/Day 8 pre-dose (“C1D8 PRE”). As can be seen inFIG. 40, serum levels of AXL and GAS6 were measured at varioustime-points during and following treatment with a tartrate salt of thecompound of structure (I) in study subjects B, D, E, and G. Serum levelsof AXL and GAS6 were measured at the following times: Cycle 1/Day 1pre-dose (“C1D1 PRE”), and 2 and 24 hours post-dose (“C1D1 2” and “C1D124”, respectively) and Cycle 1/Day 8 pre-dose (“C1D8 PRE”). The dosageadministered to each subject is shown in the lower right of the figure.

FIGS. 37-40 show results of various immunohistochemistry stains againsthuman patient primary and secondary (metastatic) tumor samples to assessepithelial-to-mesenchymal transition (EMT) markers measured at varioustime-points during disease progression. FIG. 37 shows immunostaining forAXL in primary (left) and secondary (right) tumor samples from breastcancer patients X and Y. FIG. 38 shows immuno staining for E-Cadherin inprimary (left) and secondary (right) tumor samples from breast cancerpatients X and Y. FIG. 39 shows immuno staining for N-Cadherin inprimary (left) and secondary (right) tumor samples from breast cancerpatients X and Y. FIG. 40 shows immuno staining for phosphorylated AXL(“pAXL”) in primary (left) and secondary (right) tumor samples frombreast cancer patients X and Y.

FIG. 41 shows plasma levels of AXL and GAS6 at day twenty (collectedfour hours post-dosing) of treatment with a tartrate salt of thecompound of structure (I), in a patient-derived xenograft model ofcolorectal cancer. Plasma AXL and GAS6 levels were reduced afteradministration of a tartrate salt of a compound of structure (I) at aconcentration of 80 mg/kg.

Expression levels of mRNA of epithelial-to-mesenchymal transitionmarkers were measured at day 20 (see FIG. 42) and day 27 (one week afterthe last dosing, see FIG. 43) of treatment with a tartrate salt of thecompound of structure (I), in a patient-derived xenograft model ofcolorectal cancer. At day 20, Snail expression was significantly reduced(p-value≤0.05, paired t-test) after administration of a tartrate salt ofa compound of structure (I) at a concentration of 80 mg/kg. At day 27,GAS6 expression was significantly increased (p-value≤0.05, pairedt-test) after treatment with either 40 mg/kg or 80 mg/kg of a tartratesalt of a compound of structure (I).

FIG. 44 shows mRNA expression level differences of markers of activateddendritic cells (CD86 and CD11c), at day 20 (top panel) and day 27 (oneweek after the last dosing, bottom panel) of treatment with a tartratesalt of a compound of structure (I), in a patient-derived xenograftmodel of colorectal cancer.

Example 14: Phase I/II Study of Tartrate Salts of Compound of Structure(I) in Subjects with Previously-Treated CFF

This is a combined Phase 1/2 study of tartrate salts of a compound ofstructure (I) that is orally administered in subjects with previouslytreated CFF/SFF. In both Phase 1 and Phase 2, study participants will beassigned to one of two defined patient groups:

-   -   Group 1 (a tartrate salt of a compound of structure (I)        monotherapy): Patients with CFF/SFF who are intolerant to, or        have progressed on, B-cell receptor antagonists and/or BCF-2        antagonists    -   Group 2 (a tartrate salt of a compound of structure (I) and        ibrutinib combination therapy): Patients with CFF/SFF who have        progressed on ibrutinib yet the treating provider considers        continuation of ibrutinib therapy to be in the best interest of        the patient.

Both groups of patients will be treated identically with a tartrate saltof a compound of structure (I) and will undergo the same studyassessments. Patient inclusion criteria include an Eastern CooperativeOncology Group (ECOG) performance status of ≤2.

Phase 1

Patients will be enrolled in Group 1 and Group 2 in cohorts of 3 to 6patients simultaneously. Group 2 will start at one dose level below theGroup 1 starting dose. In each group, escalation of the dose of atartrate salt of a compound of structure (I) will follow a standard 3+3design with sequential cohorts of three patients treated withincrementally higher doses of a tartrate salt of a compound of structure(I) until a dose-limiting toxicity (DLT) is observed and the maximumtolerated dose (MTD) is established. In the absence of DLTs, the dosewill be increased using a modified Fibonacci dose escalation scheme.

Once the MTD or preliminary RP2D is identified, an expansion cohort ofup to 6 patients will be enrolled in each patient group to confirmsafety/suitability of the preliminary RP2D, to collect additionalbiomarker data, and to further explore efficacy.

It is expected that up to 27 patients will be enrolled in each patientgroup for a total of up to 54 patients (a tartrate salt of a compound ofstructure (I) monotherapy and combination therapy with ibrutinib).

Additional dose levels, schedules, or disease indications of a tartratesalt of a compound of structure (I) may be explored, as appropriate,based on the modulation of key biomarkers and the safety profile andclinical signals of activity.

Monotherapy—Group 1: A tartrate salt of a compound of structure (I) willbe administered in a flat dose based on the dose in the current Phase 1of a solid tumor study at the time of initiation of this study. It issuspected that the dose will be between 33 mg and 45 mg. The study drugwill be administered orally once daily for 28 days (each cycle is 28days; no drug-free period). Patients may continue to receive a tartratesalt of a compound of structure (I) in 28-day cycles at the same dosegiven during Cycle 1 until they experience unacceptable toxicity orunequivocal disease progression. No intrapatient escalation of the doseof a tartrate salt of the compound of structure (I) is permitted.

Combination therapy—Group 2: A tartrate salt of a compound of structure(I) and ibrutinib combination therapy: The starting dose of a tartratesalt of the compound of structure (I) will be one dose level lower thanthe Group 1 starting dose, administered orally once daily for 28 days(each cycle is 28 days; no drug-free period). Patients will also receiveibrutinib at the same dose that they were receiving immediately prior tostudy enrollment. Patients should continue with the combination ofibrutinib and a tartrate salt of a compound of structure (I) for atleast 3 months after study start.

Phase 2

In Phase 2, patients will be enrolled in Group 1 (a tartrate salt of acompound of structure (I) monotherapy) and Group 2 (a tartrate salt of acompound of structure (I) combination therapy with ibrutinib) based onthe Simon 2 stage design. In Stage 1, up to 13 patients will be enrolledinto each patient group (total of 26 patients). If there are noresponses among these 13 patients in each group, the study will bestopped. Otherwise, Stage 2 will open to enroll 14 additional patientsin each group for a total of 27 patients per group. If 4 or moreresponses are observed among 27 patients, the conclusion will be thatthe study treatment is worthy of further investigation.

If both patient groups enroll through Stage 2, it is anticipated thatthe total enrollment for Phase 2 will be 54 patients.

Monotherapy—Group 1: The starting dose of a tartrate salt of a compoundof structure (I) will be the RP2D determined during Phase 1. Thetartrate salt of the compound of structure (I) will be administeredorally at a fixed dose once daily for 28 days (each cycle is 28 days; nodrug-free period) with repeated cycles permitted until a patientexperiences unacceptable toxicity or unequivocal disease progression.

Combination therapy—Group 2: The starting dose of a tartrate salt of acompound of structure (I) will be the RP2D determined during Phase 1.Patients will also receive ibrutinib at the same dose that they werereceiving immediately prior to study enrollment. Both a tartrate salt ofa compound of structure (I) and ibrutinib will be administered orally atfixed doses once daily for 28 days (each cycle is 28 days; no drug-freeperiod).

Example 15: Pre-Treatment AXL and GAS6 Levels as Predictors of DiseaseProgression

A phase 1a/1b, first-in-human, open-label, dose-escalation, safety,pharmacokinetics, and pharmacodynamic study is underway for treatmentadvanced solid tumors using a tartrate salt of a compound of structure(I). The compound is administered once daily for the first 21 out of 28days. For Phase 1a (dose escalation), sequential cohorts of three (3)patients are treated with escalated doses until the MTD is established.In the absence of dose-limiting toxicities (DLTs), the doses areincreased using a modified Fibonacci dose escalation scheme.

Blood samples were collected prior to drug treatment (baseline) andprocessed for serum. Baseline serum samples were analyzed for solubleAXL and GAS6 using the Biotechne (R&D Systems) Ella platformmicrofluidic ELISA. Subjects were treated with 1.5-28 mg/m² of atartrate salt of a compound of structure (I) for 21 consecutive daysfollowed by a 7 day treatment holiday. Subjects with documented diseaseassessment (Progressive Disease (PD) or Stable Disease (SD)) were usedto compare baseline levels of the analyses. A Wilcoxon rank sum test wasused to test the alternative hypothesis (PD<SD) for both AXL and GAS6.

FIGS. 45A and 45B show a graph of sAXL protein measured from patientswho had PD versus SD during the study. FIG. 45A contains data from 17patients (9 PD and 8 SD patients), and FIG. 45B contains data from anadditional 2 PD patients and an additional 1 SD patient (for a total of20 patients). This result indicates that patients whose diseaseprogressed during the treatment with a tartrate salt of the compound ofstructure (I) had significantly lower levels of sAXL protein prior totreatment, as compared to patients who developed SD during thetreatment. FIGS. 46A and 46B show a graph of sGAS6 protein measured frompatients who had PD versus SD during the study. FIG. 46A contains datafrom 17 patients (9 PD and 8 SD patients), and FIG. 45B contains datafrom an additional 2 PD patients and an additional 1 SD patient (for atotal of 20 patients). This result indicates that patients whose diseaseprogressed during the treatment with a tartrate salt of the compound ofstructure (I) had, on average, lower levels of sGAS6 protein prior totreatment, as compared to patients who developed stable disease duringthe treatment. In summary, both soluble AXL and GAS6 were elevated atbaseline in subjects that demonstrated a best response of SD vs PD.

For the Phase 1b expansion study, five groups are enrolled on a flatdosing schedule. Each group includes 20 patients and 10 biopsies will becollected. The study groups include: (1) Progression on Immunotherapy(combined with immunotherapy): (2) EGFR⁺ Non-small cell lung cancer,Progression after <2 tyrosine kinase inhibitors (TKIs) (combined with aTKI inhibitor); (3) Colorectal cancer, BRAF/KRAS/NRAS mutated; (4)Ovarian cancer, persistent/recurrent (platinum Resistant/Refractory);and (5) Melanoma, BRAF⁻ mutated.

Example 16: Immune Response Modulation by an AXL Kinase Inhibitor

To assess the effects of AXL inhibition on the immunosuppressiveregulatory T cells (Treg), induced Tregs were treated with a tartratesalt of a compound of structure (I) and both cell viability and cytokinerelease following treatment were assayed. Treg differentiation wasinduced from a pooled peripheral blood mononuclear cell (PBMC) sample,using the CellXVivo Human Treg cell differentiation kit (R&D Systems)according to manufacturer protocol, and confirmed differentiation usingqPCR for FoxP3 (FIG. 47A). iTregs were then treated with a tartrate saltof the compound of structure (I) at the concentrations indicated (FIG.47B). iTregs exhibited a cell viability EC₅₀ of 245 nM with treatmentwith a tartrate salt of a compound of structure (I). Using the Luminexassay platform, multiple Treg markers were assessed following treatmentwith a tartrate salt of a compound of structure (I) (FIG. 47C). Multiplemarkers were observed to increase following treatment at concentrationsup to 25 μM. To assess the effects of treatment with a tartrate salt ofa compound of structure (I) on tumor immune cell infiltration, thepresence of gene expression signatures representing multiple immune celltypes were measured in 4T1 syngeneic breast cancer model tumors. Markersof infiltrating immune cells were assessed on both formalin-fixed andfresh tissues using standard immunohistochemical and real-time PCRtechniques. Shown in FIGS. 48-54 are graphical assessments of the immunecell types found in samples treated with vehicle or 25 mg/kg of atartrate salt of a compound of structure (I). FIG. 48 shows total tumorinfiltrating lymphocytes (TILs), FIG. 49 shows dendritic cells, FIG. 50shows macrophages, FIG. 51 shows neutrophils, FIG. 52 shows naturalkiller (NK) cells, FIG. 53 shows regulatory T cells (Tregs), and FIG. 54shows exhausted CD8 T cells.

Tumor samples were harvested at study termination. While a decrease intotal tumor infiltrating lymphocytes (TILs) was observed, increaseddendritic cell infiltration and a concomitant decrease in theimmunosuppressive Tregs were also observed. These results indicate thatthe immune response affected by a tartrate salt of the compound ofstructure (I) is associated with dose-related increases in the percentof tumor-infiltrating effector CD4+ and CD8+ T cells and enhancedtherapy responses to immune checkpoint inhibitors. In addition,treatment with a compound of structure (I) results in an increase inactivated dendritic cells and a reduction in immune-suppressiveinfiltrating neutrophils and regulatory T-cells. Taken together, thesepreclinical data support the potential therapeutic activity of acompound of structure (I) as an immune modulating agent capable ofenhancing tumor immune response as a single agent and when combined withtherapies targeting immune checkpoints.

Example 17: Anti-Tumor Effects of the Compound of Structure (I)

The anti-tumor effects of tartrate salts of the compound of structure(I) have been tested in several mouse models of solid tumors. Table 10below summarizes the % TGI of a tartrate salt of the compound ofstructure (I) in several mouse xenograft models, either alone, or incombination with other therapies.

TABLE 10 Single and combination activity of a tartrate salt of acompound of structure (I) in several xenograft models Model Activity [%TGI (dose level)] KRAS mutant colorectal  98.8(90 mg/kg) cancer(HCT-116) Colorectal cancer PDX  65.3(80 mg/kg) Acute myeloid leukemia(MV-4-11) 108.0(80 mg/kg) Lung cancer (A549) 111.1(80 mg/kg) Lung cancer(A549,  88.0(75 mg/kg) combination with Erlotinib) Lung cancer (H1650,139.3(25 mg/kg) combination with Erlotinib) Lung cancer (H1650, 130.3(40mg/kg) combination with Osimertinib)

Example 18: Immuno-Oncology Effects of the Compound of Structure (I), ina Syngeneic Breast Cancer Model

In this study, tartrate salts of the compound of structure (I) wasevaluated in combination with an immune checkpoint inhibitor (ICB) in anICB-resistant triple negative breast cancer mouse model (4T1). In the4T1 syngeneic model, anti-PD-1 monotherapy did not inhibit tumor growthindicating that this tumor is resistant to anti-PD-1 treatment, as shownin FIG. 55. On the other hand, the combination of a tartrate salt of thecompound of structure (I) and anti-PD-1 resulted in statisticallysignificant tumor growth inhibition versus monotherapy with a tartratesalt of the compound of structure (I) (p<0.05). The combination effectwas influenced by potential CD8+ T cell depletion (see FIG. 56A and FIG.56B). However, the effects of a tartrate salt of the compound ofstructure (I) were further investigated on immune cells in spleen andtumors, which showed the tumor growth inhibition was associated withsignificant decreases in myeloid-derived suppressor cells (MDSC) in thespleen (see FIG. 57) and an increase in infiltration and activation ofdendritic cells (DCs) in the tumor (see FIGS. 58A and 58B). Geneexpression analysis revealed that treatment with a tartrate salt of thecompound of structure (I) decreased multiple immunosuppressive cytokinesand chemokines including IL-6 and G-CSF in vivo (see FIG. 59 and FIG.60). These results indicate that the compound of structure (I) modulatesthe immune-suppressive tumor microenvironment (TME) to reinvigorate Tcell immunity in anti-PD-1 resistant 4T1 tumors. In conclusion, that AXLinhibition with the compound of structure (I) modulates TME and enhancesthe effects of ICBs in an anti-PD-1 resistant mice tumor model.

Example 19: The Compound of Structure (I) is Active in Pre-ClinicalModels of EGFR Positive Non-Small Cell Lung Cancer

It was hypothesized that treatment with a tartrate salt of the compoundof structure (I) may potentiate EGFRi treatment in cancer, and inparticular EGFR mutant NSCLC. To interrogate this hypothesis, cell weretreated with a tartrate salt of the compound of structure (I) and cellviability was assessed with the Celltiter-Glo assay, changes in mRNAexpression were assayed using RT-qPCR, and protein expression changeswere assayed using standard immunoblotting.

FIGS. 110A-110B shows EMT marker expression in the compound of structure(I)-treated NSCLC cells. H1650 (FIG. 110A) and A549 cells (FIG. 110B)were treated for two hours with a tartrate salt of the compound ofstructure (I) at concentrations up to 2 μM, following which snail andslug mRNA expression was assessed using standard qPCR technique.

FIGS. 111A-111B show EMT marker protein expression in AXLinhibitor-treated H1650 and A549 cells. H1650 cells were treated with0.1, 0.5, or 1.0 μM of a tartrate salt of the compound of structure (I)or R428, for 24 hours, following which cells were harvested andE-cadherin and snail protein expression was assessed using standardwestern immunblotting technique (FIG. 111A). A549 cells were treatedwith 0.1, 0.5, or 1.0 μM of a tartrate salt of compound of structure (I)or R428, for 24 hours, following which cells were harvested andE-cadherin and Snail protein expression was assessed using standardwestern immunblotting technique (FIG. 111B).

In mRNA and protein assays, observed changes were consistent with areversal of the mesenchymal phenotype. Following treatment, Slug mRNAexpression was inhibited as much as 3.8-fold. However, E-cadherinexpression was increased by 1.6-fold.

To assess the combination in vivo, the H1650 xenograft model for NSCFCwas utilized. In pharmacodynamic assessment of EMT markers in vivo, asmuch as a 56% reduction in Snail protein expression was observedfollowing a single dose of a tartrate salt of the compound of structure(I) (40 mpk, at 24 hrs). FIG. 112 shows slug mRNA expression in atartrate salt of the compound of structure (I) treated H1650 xenograftmice. H1650 xenograft tumor bearing mice were treated with a tartratesalt of the compound of structure (I) (40 mg/kg) by oral gavage,following which tumors were harvested at varying timepoints followingdosing. Slug and E-cadherin mRNA expression was assessed by standardqPCR technique. FIG. 113 shows snail protein expression in the compoundof structure (I)-treated H1650 xenograft mice. H1650 xenograft tumorbearing mice were treated with a tartrate salt of the compound ofstructure (I) (40 mg/kg) by oral gavage, following which tumors wereharvested at varying timepoints following dosing. Slug and E-cadherinprotein expression was assessed by standard immunoblotting technique.

In cell viability assays in the H1650 NSCLC cell line, a tartrate saltof the compound of structure (I) showed an EC₅₀ of 39 nM, whileosimertinib showed an EC₅₀ of 2.2 μM. FIG. 18C and FIG. 18D show atartrate salt of the compound of structure (I) and EGFRi activity in theH1650 NSCLC cell line. H1650 cells were incubated in the presence of theindicated drugs for 72 hours, following which cell viability wasassessed using the CellTiter-Glo reagent according to manufacturerprotocol. H1650 cells treated with single-agents (IC₅₀): a tartrate saltof the compound of structure (I) (35.9 nM), erlotinib (9.9 μM), orosimertinib (2.2 μM) (FIG. 18C). H1650 cells were also treated withcombinations of a tartrate salt of the compound of structure (I) anderlotinib (FIG. 18D).

In assessment of treatment efficacy in vivo, and with treatment with atartrate salt of the compound of structure (I) (40 mpk, qd), 60% tumorgrowth inhibition (% TGI) was opbserved over the course of a 21-daytreatment regimen. FIG. 18E and FIG. 18F also show combination activityof a tartrate salt of the compound of structure (I) and erlotinib in theH1650 xenograft model. H1650 xenograft tumor bearing mice were treateddaily by oral gavage with either a tartrate salt of the compound ofstructure (I) (40 mg/kg), erlotinib (20 mpk), or the combination. Tumorvolumes and bodyweights were assessed twice weekly.

With osimertinib treatment (20 mpk, qd), 121% TGI was observed. However,with the combination, 140% TGI was observed. FIG. 18G and FIG. 18H showcombination activity of a tartrate salt of the compound of structure (I)and osimertinib in the H1650 xenograft model. H1650 xenograft tumorbearing mice were treated daily by oral gavage with either a tartratesalt of the compound of structure (I) (40 mg/kg), osimertinib (20 mpk),or the combination. Tumor volumes and bodyweights were assessed twiceweekly. Due to its ability to reverse the aggressive mesenchymalphenotype of cancer cells, the compound of structure (I) is a promisingagent with the potential to have single agent activity and combinedsynergy with targeted anti-cancer agents.

Example 20: The Compound of Structure (I) Demonstrates Efficacy inPreclinical Models of Colorectal Cancer Independent of KRAS MutationStatus

Tartrate salts of the compound of structure (I) is evaluated foractivity against colorectal cancer (CRC). In cell viability assays ofCRC lines, treatment with a tartrate salt of the compound of structure(I) resulted in IC₅₀ values ranging from 4.5-123 nM. Notably, cellgrowth inhibition following treatment with a tartrate salt of thecompound of structure (I) was independent of KRAS mutation status; theKRAS mutant HCT-116 line was the most sensitive CRC cell line tested.FIG. 104A shows KRAS mutation status of selected CRC cell lines. FIG.104B shows CRC cell viability determination following 72 hrs treatmentwith a tartrate salt of the compound of structure (I) and assessment viaCellTiter-Glo.

Mesenchymal markers, including Snail, were suppressed by 7.6-fold (mRNA)and 4.9-fold (protein) in the HCT-116 line at 500 nM. For example, FIG.105A and FIG. 105B show that a tartrate salt of the compound ofstructure (I) suppresses mesenchymal markers without modulatingepithelial markers. HCT-116 cells were treated with indicatedconcentrations of AXL inhibitors: R428, RXDX-106, and a tartrate salt ofthe compound of structure (I) for 24 hrs. FIG. 105A shows mRNAexpression levels were quantified via RT-qPCR. FIG. 105B shows proteinexpression levels were analyzed via western blot. Snail expression wassuppressed by 7.6 fold (m-RNA) and 4.9 fold (protein) with 500 nM of atartrate salt of compound of structure (I).

Activity of a tartrate salt of the compound of structure (I) was alsoassessed in vivo using two KRAS mutant CRC models: HCT-116 and apatient-derived xenograft (PDX) model. In the HCT-116 xenograft model,single agent treatment with a tartrate salt of the compound of structure(I) achieved 69% tumor growth inhibition (% TGI) with an oral dosingschedule at 40 mg/kg. Athymic nude mice were injected in the hind-flankwith 10 million cells and stratified into cohorts of 10 mice. Compoundswere formulated in 5% (w/v) TPGS and 1% (v/v) PS80 in H20 andadministered my oral gavage. Tumor volumes (FIG. 106A) and body weightswere measured twice a week (FIG. 106B). Intra-tumoral GAS6 expressionwas quantified via RT-qPCR. The 40 mg/kg of a tartrate salt of compoundof structure (I) cohort achieved 69% TGI without adverse events (FIG.106C).

In a KRAS-mutant PDX model, a tartrate salt of the compound of structure(I) achieved 44% TGI when mice were dosed at 40 mg/kg. Balb/c nude micewere implanted with 2-3 mm fragments of primary human CRC tumors andthen stratified into cohorts of 10 mice. A tartrate salt of the compoundof structure (I) was formulated in 5% (w/v) TPGS and 1% (v/v) PS80 inH20 and administered by oral gavage. Tumor volume (FIG. 107A) andbodyweights (FIG. 107B) were measured twice a week. The 40 mg/kg of atartrate salt of a compound of structure (I) cohortachieved 44% TGIwithout adverse events.

Pharmacodynamic analyses were performed on tissues from the HCT-116 andPDX models. The ligand for AXL, GAS6, was significantly upregulated intissues after treatment with a tartrate salt of the compound ofstructure (I) in both CRC in vivo models while soluble AXL and GAS6 weresignificantly downregulated in plasma in the PDX model. Furthermore,Axin2, a Wnt/βcatenin regulated gene, was significantly downregulated bythe compound of structure (I) in tumor tissue from the PDX model,suggesting inhibition of the Wnt/βcatenin pathway. These data support apotential role for AXL in the promotion of the mesenchymal phenotype inCRC and show that AXL inhibition by a tartrate salt of the compound ofstructure (I) suppresses the mesenchymal phenotype and is effectiveagainst CRC cells regardless of KRAS mutation status.

FIGS. 108A-108E show a tartrate salt of the compound of structure (I)suppresses sAXL/sGAS6 concentrations while down-regulating Wnt/β-cateninregulated genesAxin2/CCND1 in the KRAS mutant PDX CRC model. sAXL (FIG.108A) and sGAS6 (FIG. 108B) were quantified in the serum via ELISA.Intra-tumoral GAS6 (FIG. 108C), Axin2 (FIG. 108D), and CCND1 (FIG. 108E)were quantified via RT-qPCR. Analyses were performed on mice treated for27 days (except Axin2; 21 days). Suppression of sAXL and sGAS6 indicatea reversal of EMT. Compound of structure (I)-mediated downregulation ofWnt/β-catenin associated genes further supports a previously reportedrole for AXL in β-catenin stabilization.

FIG. 109 shows a positive correlation between sAXL/sGAS6 and tumorvolume in the KRAS mutant PDX CRC model identifies them as potentialbiomarkers for disease progression. Soluble concentrations werequantified in each mouse's serum via ELISA followed linear regressionanalysis. Statistical significance for each correlation: sAXL and tumorvolume (P<0.005); and sGAS6 and tumor volume (P<0.0005).

Example 21: Anti-Tumor Effects of the Compound of Structure (I) onInflammatory Breast Cancer

Tartrate salts of the compound of structure (I) is evaluated foractivity against inflammatory breast cancer in cell culture and in axenograft model.

The activity of a tartrate salt of the compound of structure (I) isevaluated in inflammatory breast cancer cell lines. Cell viability isassessed (e.g., using a CellTiter Glo assay) at a range of doses, and anIC₅₀ value is determined. Examples of inflammatory breast cancer celllines that may be used include SUM149, KPL-4, and SUM190.

The effects of a tartrate salt of the compound of structure (I) areassessed in an inflammatory breast cancer xenograft model. An example ofan inflammatory breast cancer xenograft model is described in Wang etal., 2013 Cancer Research 73(21): 6516-6525. Athymic BALB/c nu/nu miceare injected into the mammary fat pads with SUM149 cells (2×10⁶ cellwith 50% Matrigel) and tumors are established. Mice are treated with atartrate salt of a compound of structure (I) or a vehicle control, forat least two weeks. Tumor volume and body weight are monitored duringthe course of treatment.

Example 22: Polymorphs of Tartrate Salts of Compound of Structure (I)

The polymorphs of the present disclosure can be prepared in view of thenovel methods, reaction schemes and examples provided herein (see, e.g.,Example 23), together with synthetic methods known in the art ofsynthetic organic chemistry, or by variations thereon as appreciated bythose skilled in the art. The reactions are performed in a solvent orsolvent mixture appropriate to the reagents and materials employed andsuitable for the transformations being effected. It will be understoodby those skilled in the art of organic synthesis that the functionalitypresent on the molecule should be consistent with the transformationsproposed. This will sometimes require a judgment to modify the order ofthe synthetic steps or to select one particular process scheme overanother in order to obtain a desired compound or polymorph of thedisclosure

The starting materials are generally available from commercial sourcessuch as Sigma Aldrich or other commercial vendors, or are prepared asdescribed in this disclosure, or are readily prepared using methods wellknown to those skilled in the art (e.g., prepared by methods generallydescribed in Louis F. Fieser and Mary Fieser, Reagents for OrganicSynthesis, v. 1-19, Wiley, New York (1967-1999 ed.), Larock, R. C.,Comprehensive Organic Transformations, 2^(nd)-ed., Wiley-VCH Weinheim,Germany (1999), or Beilsteins Handbuch der organischen Chemie, 4, Aufl.ed. Springer-Verlag, Berlin, including supplements (also available viathe Beilstein online database)).

In the preparation of the polymorphs of the compound of structure (I),protection of remote functionality of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods. Theneed for such protection is readily determined by one skilled in theart. For a general description of protecting groups and their use, seeGreene, T. W. et al., Protecting Groups in Organic Synthesis, 4th Ed.,Wiley (2007).

Additionally, the polymorphs of present disclosure exhibit valuablepharmacological properties, which can be demonstrated at least by usingany one of the following test procedures. Accordingly, polymorphs of thepresent disclosure were assessed in biochemical assays as set forthbelow. Data was acquired according to the parameters listed below:

X-Ray Powder Diffraction (XRPD):

PANalytical XRPD instrument. The solid sample was spread on azero-background Si sample holder. The XRPD parameters used are listedbelow in Table 11:

TABLE 11 Settings for acquiring XRPD data Parameter Value for DataAcquisition X-Ray Wavelength Cu, kα Kα1 (Å): 1.540598 Kα2: (Å): 1.544426Kα2/Kα1 intensity ratio: 0.50 X-ray tube setting 45 kV, 40 mA Divergenceslit ⅛° Scan mode continuous Scan range (°2θ) 3°-40° Step Size (°2θ)0.263 Scan step time (s) 50 Test time(s) ~5 minutes 4 secondsTGA and DSC:

TGA data were collected using a TA 5500 TGA from TA Instruments and DSCwas performed using a TA 2500 DSC from TA Instruments. Detailedparameters used are listed in Table 12 below.

TABLE 12 Instrument settings for acquiring TGA and DSC data ParametersTGA DSC Method Ramp Ramp Sample pan Aluminum, open Aluminum, crimpedTemperature RT - desired temperature 25° C. - desired temperatureHeating rate 10° C./min 10° C./min Purge gas N₂ N₂HPLC:

Agilent 1260 HPLC was utilized and detailed chromatographic conditionsfor purity and solubility measurement are listed in Table 13 below.

TABLE 13 HPLC settings for acquiring data Parameters Value HPLC Agilent1260 with DAD detector Column Ascentis Express C18, 4.6 mm × 100 mm, 2.7μm Mobile phase A: 0.1% H₃PO₄ in H₂O B: Acetonitrile Time (min) % BGradient table 0.0 10 6.0 95 8.0 95 8.1 10 10.0 10 Run time 10.0 minPost time 0.0 min Flow rate 1.5 mL/min Injection volume 10 μL Detectorwavelength UV at 210 nm Column temperature 40° C. Sampler temperatureambient Diluent acetonitrile:H₂O (1:1, v/v)

Example 23: Preparation of Crystalline Form A

Compound 1A was obtained according to methods known in the art and metpurity specifications, however, ¹H-NMR analysis showed that the materialincluded about 30% of triethylamine, which was used in in a previoussynthetic step. Applicant discovered that re-slurry in hot ethanolefficiently removed triethylamine. The re-slurry step is performed asfollows:

A mixture of 1A and ethanol was heated at 70° C. for 2 hours and thenslowly cooled to 20° C. over 5 hours. The slurry was filtered and driedunder vacuum to provide purified 1B. This isolated crystal was subjectedto a reprocessing procedure to provide 1C.

The reprocessing procedure included dissolving 1C in a chloroform andethanol mixture and activated charcoal was added. The resulting slurrywas stirred at room temperature for 1 hour and filtered. The filteredsolid was washed, combined with filtrate and the solvents were removedby distillation. Then ethanol was added, and distillation repeated toremove chloroform. After the distillation, resulting slurry was cooledand filtered to give purified 1C. The obtained material was dissolvedwith mixture of anisole and ethanol at 70° C. Ethanol solution oftartaric acid was then added to this solution and subsequently seededwith Form A. The resulting slurry is cooled to 20° C. and filtered,washed with ethanol, dried to afford the polymorph of a tartaric acidsalt of a compound of structure (I). The purity of the desired productwas assessed to be 99.5% by HPLC.

¹H NMR (400 MHz, DMSO-d6) δ=9.56 (s, 1H), 9.33 (s, 1H), 8.58 (s, J=8.0Hz, 1H), 8.29 (s, 1H), 7.83 (dd, J=8.0, 1.6 Hz, 1H), 7.71 (td, J=7.2,1.4 Hz), 7.57 (d, J=8.4 Hz, 2H), 7.38 (td, J=7.2 Hz, J=1.0 Hz), 7.18 (d,J=8.4 Hz, 2H), 4.19 (s, 4H), 3.51 (s, 2H), 2.86 (bs, 3H), 2.65 (s, 6H),2.60-2.50 (m, 8H)

Proton signals at 4.19 and 3.51 ppm correspond to tartaric acid. Basedon the integration of those peaks, tartaric acid to compound ofstructure (I) was consistently 2:1.

Example 23A: Preparation of Crystalline Form A

2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide(5 g, 9.69 mmol) was dissolved in anisole (75 g) and EtOH (30 g) at 70°C. Tartaric acid (2.91 g, 19.38 mmol) dissolved in EtOH (30 g) was addedto the mixture over 1 h, then small amount of seed crystal* was added tothe solution to initiate the precipitation. The mixture was stirred for1 h and cooled to 20° C. over 5 h. The solid was collected byfiltration, washed with EtOH and dried to afford2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidedi-tartrate salt as a white solid (7.35 g, 9.01 mmol). * Note: Seedcrystals of Form A were obtained by combining2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamideand tartaric acid in anisole/ethanol under the conditions specifiedabove, followed by initiation of crystal formation using one or moretechniques such as (1) cooling the solution (e.g., to room temperature,or in a freezer), (2) concentrating the solution (e.g., by slowevaporation, or with a rotart evaporator), and/or (3) scratching theinterior of the flask containing the solution. Crystals obtained in thismanner were confirmed to be of Form A by ¹HNMR and XRPD analysis.

The purity of the desired product was assessed to be 99.5% by HPLC.

¹H NMR (400 MHz, DMSO-d6) δ=9.56 (s, 1H), 9.33 (s, 1H), 8.58 (s, J=8.0Hz, 1H), 8.29 (s, 1H), 7.83 (dd, J=8.0, 1.6 Hz, 1H), 7.71 (td, J=7.2,1.4 Hz), 7.57 (d, J=8.4 Hz, 2H), 7.38 (td, J=7.2 Hz, J=1.0 Hz), 7.18 (d,J=8.4 Hz, 2H), 4.19 (s, 4H), 3.51 (s, 2H), 2.86 (bs, 3H), 2.65 (s, 6H),2.60-2.50 (m, 8H)

Proton signals at 4.19 and 3.51 ppm correspond to tartaric acid. Basedon the integration of those peaks, tartaric acid to compound ofstructure (I) was consistently 2:1.

Example 23B: Preparation of Crystalline Form B

2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidemono-tartaric acid salt (1.75 kg, 2.62 mol) was added 5.25 L of EtOH and4.0 L of solvent distilled under reduced pressure. This was repeatedtwice until the water content by Karl Fisher Titration is below 0.1%.Then 52.5 L of Ethanol and heated to reflux. Then 0.513 kg ofL-(+)-tartaric acid in 17.5 L of ethanol was added slowly over 2 hoursand seeded at 70° C. Then stirring was kept for 12 hours at 70° C. andagain heated to 80° C. and kept for 2 hours. The slurry was cooled to20° C. over 6 hours, and then kept stirring for additional 12 hours. Thecrystals were filtered and then washed with 3.5 L of Ethanol twice. Thecrystal was dried under reduced pressure to afford 1.8 kg of crystal.Then 1.6 kg of this material was suspended with 16.0 L of methyl t-butylether and 0.30 kg of bis(pinacolate) diboron was added at 30° C. Thenthe mixture was heated at 40-45° C. for 12 hours and cooled to 30° C.and filtered. The filtered solid was washed 8.2 L of methyl t-butylether and dried under vacuum to afford2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidemono-tartaric acid salt (1.38 kg, 2.07 mol).

Example 23C: Preparation of Crystalline Form D

2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide(5 g, 9.69 mmol) was charged into EtOH (119 g) and the mixture washeated to 80° C. Tartaric acid (1.45 g, 9.69 mmol) dissolved in EtOH (40g) was added to the mixture over 2 h at the same temperature, then themixture was cooled to 70° C. before small amount of seed crystal* wasadded to initiate the precipitation. The mixture was stirred for 2 h andcooled to 20° C. over 5 h. The solid was collected by filtration, washedwith EtOH and dried to afford2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamidemono-tartrate salt as a white solid (5.98 g, 8.98 mmol). *Note: Seedcrystals of Form D were obtained by combining2-((5-chloro-2-((4-((4-methylpiperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamideand tartaric acid in anisole/ethanol under the conditions specifiedabove, followed by initiation of crystal formation using one or moretechniques such as (1) cooling the solution (e.g., to room temperature,or in a freezer), (2) concentrating the solution (e.g., by slowevaporation, or with a rotart evaporator), and/or (3) scratching theinterior of the flask containing the solution. Crystals obtained in thismanner were confirmed to be of Form D by ¹HNMR and XRPD analysis.

Example 24: Characterization of Various Polymorphic Forms of TartrateSalts of Compound of Structure (I)

(i) Stability and Reproducibility

Other polymorphic forms of the compound of structure (I) were comparedfor their relative stability under different storage conditions. Otherpolymorphic forms include the free base form of a compound of structure(I), Form B, and Form D. The XRPD diffractogram of Forms B and D areshown in FIGS. 62 and 63, respectively.

It was discovered that Form A is a di-tartaric acid salt form and it wasidentified as having only one crystal form (Form A). For comparison,XPRD patterns showing an overlay of Form A (lower) and Form B (upper)illustrates a similar pattern, with some additional peaks.

(ii) Stability Profiles

The different forms of the compound of structure (I) (i.e., FB, Form A,Form B, and Form D were each stored at 40° C. and a relative humidity of75%. After 3 weeks, all samples showed good chemical stability with nosignificant HPLC purity decrease. Form change was observed only for FormB. Additionally, as shown in FIG. 65, which compares Form A and Form B,Form B appears to have weaker peak intensities. The weaker peakintensity can possibly be attributed to lower crystallinity.

(iii) Physical and Chemical Properties

The table below summarizes the characterization and solid statestability results observed for FB and Forms A, B, and D. Samples wereprepared and data was obtained according to the procedures and processeslisted above unless otherwise specified. More procedures, processes, andresults are listed below in Table 14.

TABLE 14 Summary of physicochemical properties evaluation for Forms A,B, D, and free base Assay Description Form A Form B Form D Free BaseXRPD Crystalline Crystalline Crystalline Crystalline Weight Loss (%) 1.82.3 2.0 0.22 (160° C.) (160° C.) (160° C.) (120° C.) Endotherm (peak, °C.) 107.8, 152.1, 189.1 101.9, 140.1 79.4, 140.7 221.1  Initial HPLCPurity 99.6 98.1 98.8 98.3 (area %) HPLC Purity at 3 99.7 98.2 98.3 98.6weeks^(#) (area %) Form Change at 3 No Yes No No weeks^(#)Hygroscopicity/water 4.6%/No 7.5%/No  5.2%/Yes  Non-hygroscopic/ uptakeat 80% RH/FC 0.07%/No Morphology Fine particles with agglomerationDiamond particles Solubility* SGF >17.4/NA >6.4/NA >11.0/NA    5.7/No(mg/mL)/ FaSSIF >14.8/NA >8.9/NA 3.9/Yes 0.021/No FC FeSSIF    5.2/NA* 4.7/NA*  4.6/NA*  1.7/No pH 2.0  >6.7/NA >6.6/NA >6.8/NA      2.6/YespH 4.0   0.45/Yes  0.26/Yes 0.23/Yes   0.17/Yes pH 6.0   3.8/Yes  2.5/Yes 1.9/Yes 0.014/No pH 8.0  0.020/Yes 0.010/Yes  0.0050/Yes  0.038/No pH 10.0 0.0098/Yes  0.018/Yes  0.053/Yes  0.076/No *Thesolubility was collected at 24 hours ^(#)under 40° C./75% RH FC: Formchange NA: solid-state results not available as clear solution formedNA*: solid-state results not available as oil sample obtained.

(a) Dynamic Vapor Sorption (DVS)

DVS results showed up to 80% relative humidity at 25° C., the wateruptake was in the range of 4.6˜7.5% for Forms A, B, and D with formchange observed for Form D. That is, Form D changed to a new form notconsistent with all the four known crystal forms, and free base wasnon-hygroscopic (water uptake less than 0.2%) with no form change at theend of the test.

(b) Kinetic Solubility

Kinetic solubility was measured in bio-relevant buffers (FaSSIF, FeSSIFand SGF) at 37° C. Compared to free base sample, significant highersolubility (>5 mg/mL) was observed for Forms A, B, and D in allbiorelevant buffers. Similar solubility observations were observed forall three forms in SGF and FeSSIF, but Form D showed lower solubilitythan other Forms A and B in FaSSIF.

(c) pH Solubility

pH solubility was measured in pH 2, 4, 6, 8 and 10 buffers at 37° C. Theresults showed: i) higher solubility in pH 2 than that of in the otherpH buffers for each sample and decreased solubility in higher pH buffer,especially in alkaline media, in which the solid form of residual solidsalso changed at the end of the test for Forms A, B, and D; ii) Form Ashowed higher solubility than other polymorphs in pH 2, 4 and 6 buffers.

Example 25: Toxicokinetic and Toxicology Profiles of Tartrate Salts ofCompound of Structure (I)

Form A of the tartaric acid salt of structure (I) was developed toimprove stability and reproducibility the drug formulation. Previousstability issues have been encountered with the polymorph Form B. Toensure the new drug formulation had an acceptable toxicokinetic andtoxicology profile; studies were conducted in mice and rats comparingthree formulations:

-   -   (1) polymorph Form A    -   (2) polymorph Form B    -   (3) polymorph Form D

In each study, comparisons were made to the free base (FB) form of thecompound of structure (I). A single-dose study was conducted in rats anda 7-day repeat-dose study performed in mice.

Single Dose Toxicity Study

In this study, a comparison of the clinical effects and pharmacokineticsof the free base (FB) and tartaric acid salt polymorph forms (Forms A,B, and D) of the compound of structure (I) following a single oral dosewere evaluated in male SD rats. The FB dose levels evaluated were 20,50, and 150 mg/kg. The test articles were formulated with 1% Tween 80/5%TPGS/Sterile Water for Injection (v/v/v) and the study designed asindicated in Table 15 below:

TABLE 15 Study Design Single Dose Toxicity in Rats Dose Dosing SolutionNumber of Dose¹ Volume Concentration Animals Group (mg/kg) (mL/kg)(mg/mL) and Sex 1. Vehicle 0 10 0 3M 2. Free Base 20 10 2 6M Low Dose 3.Free Base 50 10 5 6M Mid Dose 4. Free Base 150 10 15 6M High Dose 5.Form B 20 10 2 6M Low Dose 6. Form B 50 10 5 6M Mid Dose 7. Form B 15010 15 6M High Dose 8. Form A 20 10 2 6M Low Dose 9. Form A 50 10 5 6MMid Dose 10. Form A 150 10 15 6M High Dose 11. Form D 20 10 2 6M LowDose 12. Form D 50 10 5 6M Mid Dose 13. Form D 150 10 15 6M High Dose¹Dose levels and dosing solution concentration were calculated asequivalents of FB based on correction for the tartaric acid content ofForm A, Form B and Form D by dose multiplication factors of 1.58, 1.35and 1.29, respectively.

Endpoints in the study consisted of mortality checks, clinicalobservations, body weight measurements and toxicokinetics. Following a7-day post-dose observation period, animals were euthanized, necropsiedand macroscopic observations recorded.

On the day of dosing, the vehicle and compound of structure (I)treatments≤50 mg/kg were well tolerated with the exception of onemid-dose animal administered Form B that was found dead 7.5 hourspost-dosing. In contrast, mortality was frequently observed on the dayof dosing for the high-dose group. Mortality was observed beginning at4.5 hours post-dosing, with 100% mortality observed by the end of therecovery period in the 150 mg/kg FB and Form A, B and D groups. Duringthe 7-day post-administration observation period, mortality ormoribundity was also noted in the mid-dose (50 mg/kg) groups but not inthe low-dose or vehicle groups. The incidence of mortality was similaracross different treatment groups.

Clinical signs were mostly present in rats at the mid- and high-doselevels, with only a few rats displaying clinical observations at thelow-dose level. Clinical observations noted in the high-dose groupsincluded passivity, soft feces and diarrhea, hunched back posture,piloerection, being “cool to the touch”, weight loss, anorexia, andseizures. For mid-dose animals dosed with free base and Forms A, B andD, the primary clinical signs were anorexia, emaciation, and weightloss. Additional clinical signs included soft feces, diarrhea, hunchedback posture, passivity, piloerection, being “cool to the touch,” andscruffy fur. The only clinical signs noted in low-dose groups were softfeces and weight loss in some animals. No clinical signs were observedin vehicle dosed animals. The nature and incidence of clinical signswere similar across the different treatment groups.

A dose-related reduction in body weight/body weight gain was seen acrossall test article groups. Animals in the low-dose group gained weightover the 7-day post-dose observation period but at a rate less than thevehicle control whereas body weight loss was seen in the mid- and/orhigh-dose groups.

Frequent findings upon necropsy in all rats from the high-dose groupwere of gastrointestinal origin and included thick yellowish fluid inthe small intestine, stomach full of undigested food, fluid filledcecum, and lack of formed feces; in one rat pallor of the spleen andliver and a reduction in the size of the thymus was noted. For rats inthe mid-dose groups, abnormal findings were less frequently observed.The findings were similar to the gastrointestinal observations made inthe high-dose groups. Non-gastrointestinal findings for several of themid-dose group rats included pallor of the kidneys, pallor of the spleenand liver, enlarged adrenal glands, and a reduction of thymus size ofseveral rats.

Following oral dosing with FB and Forms A, B and D, plasmaconcentrations of the compound increased in a dose-dependent manner.Comparatively, all three tartaric acid salt polymorph forms of thecompound of structure (I) displayed similar plasma concentrationprofiles. Dose proportionality or close to dose proportionality for theplasma exposure to FB based on C_(max) and AUC₀₋₄ hrs was observed forFB and Form B, while somewhat higher than dose proportional plasmaexposure was observed for Form A and Form D.

In conclusion, oral administration of FB and Forms A, B and D at singlefree base equivalent dose levels of 50 and 150 mg/kg produceddose-dependent adverse effects in male rats, mortality at the high doseand similar and significant clinical findings at the mid and high doselevels characterized by anorexia, emaciation, weight loss, soft feces,diarrhea, hunched back posture, and scruffy fur. The dose level of 20mg/kg was generally well tolerated by male rats with minor clinicalobservations and a reduction in body weight gain. Necropsy findings inrats dosed with FB and Forms A, B and D were similar but more frequentin the high dose group compared to the mid dose groups with abnormalgastrointestinal findings being the most common observation. Some higherdosed animals also displayed pallor of the liver and spleen, enlargementof the adrenal glands and a reduction in the size of the thymus. Oraladministration of FB and free base equivalents of tartaric acid saltpolymorph Forms A, B and D resulted in similar FB plasma profiles.Overall, similar exposures and dose-related adverse effects wereobserved in rats following oral dosing with Forms A, B, and D.

Repeat Dose Toxicity and Toxicokinetics

In this study, the clinical effects and the pharmacokinetics of thecompound of structure (I) free base (FB) and three tartaric acid saltpolymorph forms including Forms A, B, and D following single and 7-dayrepeated oral free base equivalent dose levels of 20, 50, and 80 mg/kgwere evaluated in CD-1 mice. The test articles were formulated with 1%Tween 80/5% TPGS/Sterile Water for Injection (v/v/v) and the studydesigned as indicated in Table 16 below:

TABLE 16 Study Design Repeat Dose Toxicity in Mice Dose Dose PKSatellite Groups Dose Dose Volume Concentration Main Day 1* Day 7**Group (mg/kg/day) (mL/kg) (mg/mL) Study (Single Dose (Repeated Dose)Control Vehicle Vehicle 0 10 0 3M^(a) — — Dosed With Different Forms ofcompound of structure (I) (i.e., Free Base, Form A, Form B and Form D)Low Dose 20 10 2 — 15M 15M Mid Dose 50 10 5 — 15M 15M High Dose 80 10 8— 15M 15M M = Male ^(a)Control animals were dosed with a vehicle for 7days *PK on Day 1 (Terminal bleeding was by cardiac puncture on 3M foreach time point, 5 time points). **PK on Day 7 following 7-day repeateddose (terminal bleeding was by cardiac puncture on 3M for each timepoint, 5 time points).

Endpoints during the study consisted of clinical observations, bodyweight, and toxicokinetics. Animals euthanized due to excessive bodyweight loss in the toxicology-designated groups were euthanized,necropsied, and macroscopic observations recorded.

Administration of a single dose of vehicle and of compound of structure(I) (free base and polymorph forms) at the low-, mid- and high-doselevels and 7 daily doses of the low- and mid-dose levels for 7 days werewell tolerated. At the high dose level only, one mouse died in the FormD group. The highest dose (80 mg/kg) resulted in a similar decline inmean body weight over the 7-day observation period across all groups. Noeffects upon body weight were seen at doses≤50 mg/kg/day.

Clinical signs were present in mice in the high-dose groups beginning onDay 6 or 7 of treatment. The clinical signs noted included passivity,lethargy, piloerection, pallor, dyspnea, hunched back posture, ptosis,pallor, prostration, coolness to the touch and weight loss. Clinicalsigns were observed in 11/15 (73%) of mice dosed with FB, 10/15 (67%),10/15 (67%), and 15/15 (100%) of mice dosed with Form A, Form B and FormD, respectively. By Day 7, severe weight loss (>25% of starting bodyweight) led to the euthanasia of 3/15 (20%) of the mice dosed with FBand 4/15 (27%), 3/15 (20%) and 4/15 (27%) of mice dosed with Form A,Form B and Form D, respectively.

Findings at early sacrifice necropsies performed for animals euthanizeddue to severe weight loss, were mostly of gastrointestinal origin. Theprimary finding was the presence of yellowish fluid in the smallintestine and in some cases, hemorrhage of the small intestine.Distension of the cecum and the presence of fluid was observed in 7animals subject to necropsy. Other organ findings included, distensionof the stomach filled with food and a reduction in the size of thethymus. The findings were generally evenly distributed across thetreatment groups.

Comparatively, all three of the polymorph forms displayed similartoxicokinetic profiles. Exposure increased with increasing dose levelswith each polymorphic form.

In conclusion, single dose oral administration of FB and Forms A, B, andD at free base equivalent doses of 20, 50 and 80 mg/kg and 7 daily dosesat 20 and 50 mg/kg/day were well tolerated. Daily administration of FBand Form A, Form B, and Form D for seven days produced clinical signsand severe body weight loss at the highest dose leading to the prematuresacrifice of several animals on Day 6 or 7 of dosing. Necropsy findingsin mice dosed that either died or were euthanized due to severe weightloss were predominantly confined to the gastrointestinal tract; areduction in thymic size was also noted. The toxicokinetic profile ofthe various polymorph formulations was similar. Overall, the nature andincidence of effects observed at the high dose in mice were similarfollowing seven days of oral dosing with FB and Forms A, B, and D.

Example 26 Ovarian Cancer Cell Lines are Susceptible to the Compound ofStructure (I)

Ovarian cancer cell lines were treated with a range of doses of atartrate salt of the compound of structure (I), and cell viability wasassayed. Cell viability at each dose was plotted to create a doseresponse curve and determine IC₅₀ values for each cell line. The IC₅₀values are shown in Table 17.

TABLE 17 IC₅₀ values Cell Line IC₅₀ Activity Ovcar3 ++ Ovcar8 ++ Skov3 +OVTOKO ++ OVMANA ++ Kuramochi ++ IC₅₀ Activity: ++ = <1 μM; + = 1 to 10μM

Example 27: Ovarian Cancer Cell Line ES-2 is Susceptible to the Compoundof Structure (I)

ES-2 cells, which is an ovarian cancer cell line of the clear cellhistotype, were assayed for viability in the presence of a tartrate saltof the compound of structure (I). Cell viability was measured at a rangeof doses of a tartrate salt of the compound of structure (I) to create adose response curve, which is shown in FIG. 66. As shown in FIG. 66, theIC₅₀ value for treating ES-2 cells with a tartrate salt of a compound ofstructure (I) is 20 nM.

Example 28: A Compound of Structure (I) Potently Inhibits the Growth ofChemotherapy Resistant Ovarian Tumor Cells and Clear Cell Ovarian TumorCells In Vitro

To examine whether a tartrate salt of a compound of structure (I) hasefficacy against chemotherapy resistant ovarian cell lines, cisplatin(CDDP) sensitivity was measured in various ovarian cell lines. PA-1,A2780, OVISE, and OVTOKO showed CDDP sensitivity (IC₅₀=<1 uM), whileother cell lines showed CDDP resistant (IC₅₀>2.5 uM). The tartrate saltof the compound of structure (I) kills several chemotherapy-resistantovarian cancer cell lines at low doses (FIG. 67, top left and rightpanels).

Furthermore, a tartrate salt of the compound of structure (I) suppressesthe growth of ovarian tumor cells more efficiently than otherAXL-targeting compounds (Cabozantinib, Foretinib) in CDDP-resistantKuramochi cell line (FIG. 67, bottom panel). Kuramochi cells weretreated with varying doses of a tartrate salt of a compound of structure(I), Cabozantinib and Foretinib for 96 hrs. Subsequently, cell viabilitywas determined by Alamar Blue assay from which IC₅₀ values for CDDP anda tartrate salt of compound of structure (I) were determined. Graphsrepresent the dose response of Kuramochi cells to each compound.Kuramochi cells were treated with the indicated concentration of atartrate salt of the compound of structure (I) or Cabozantinib for 8hrs, lysed and western blotted for tubulin and AXL phosphorylated atY779 or Y702 site. Phospho-AXL (Y702) were reduced by a tartrate salt ofthe compound of structure (I) (100 nM) as well as Cabozantinib (1 μM), ac-Met, VEGFR2, and AXL inhibitor (FIG. 68). Note the dose-dependent lossof AXL phosphorylation at Y702 site, which best represents AXL kinaseactivity, by treatment with the compound of structure (I).

Example 29: EMT Markers are Suppressed in Ovarian Cancer Cells byTreatment with the Compound of Structure (I)

Epithelial to mesenchymal (EMT) markers were quantified by mRNAexpression analysis (using RT-qPCR), following treatment with a tartratesalt of the compound of structure (I). As shown in FIG. 69, two hoursafter treatment with a tartrate salt of the compound of structure (I),EMT markers are suppressed by higher doses of the compound of structure(I).

However, as shown in FIG. 70, EMT markers are no longer suppressedtwenty-four hours after treatment with a tartrate salt of the compoundof structure (I). As shown in FIG. 71, protein expression levels weremeasured for the EMT markers Snail and Slug following treatment with atartrate salt of the compound of structure (I), and the proteinexpression levels were consistent with the mRNA expression levels.

Example 30: A Compound of Structure (I) Potently Inhibits the Migrationof Ovarian Tumor Cells

FIG. 72 shows the effects of AXL kinase inhibitors (a tartrate salt of acompound of structure (I) and BMS-777607) on the migration of ovariantumor cells in a scratch assay. Ovarian tumor cells ES-2 werepre-treated with varying doses of a tartrate salt of the compound ofstructure (I) or another AXL inhibitor, BMS-777607, for 6 hours beforescratching by the incucyte wound maker. The images were captured after18 hours post-scratching. The tartrate salt of the compound of structure(I) showed strong inhibition of migration of ES-2 cells, an ovarianclear cell carcinoma cell line, whereas another AXL inhibitor,BMS-777607, did not inhibit migration, even at 10 μM.

Example 31: In Vivo Efficacy of the Compound of Structure (I) in a MouseOvarian Cancer Xenograft Model

The efficacy of a tartrate salt of the compound of structure (I) wasmeasured using a murine ES-2 intraperitoneal xenograft model. Mice wereinoculated intraperitoneally with ES-2 cells, and then treated witheither: a vehicle (n=6), 25 mg/kg of a tartrate salt of the compound ofstructure (I) (n=6), or 50 mg/kg of a tartrate salt of the compound ofstructure (I) (n=5).

Treatment significantly suppressed ascites development (ascites volume,vehicle: 3.34 mL, a tartrate salt of a compound of structure (I): 0.28mL; abdominal circumference, vehicle: 7.96 cm (day 15), a tartrate saltof a compound of structure (I): 6.82 cm (day 15), p<0.05). FIG. 73 showsthe circumference of the mice following treatment (from day five to 15day nineteen after implant), and a representative photo of a mouse ineach treatment group on day 20 after implant. FIG. 74 shows a graph ofthe body weight of mice following treatment, from day five to daynineteen following implant. As can be seen in FIG. 73 and FIG. 74, atartrate salt of the compound of structure (I) showed efficacy at 25mg/kg and 50 mg/kg in the ES-2 intraperitoneal dissemination model.

Example 32: Cytokines and Chemokines Profiles in Ascites Fluid FollowingIn Vivo Treatment with a Compound of Structure (I)

A panel of chemokines and cytokines were measured two hours, twenty-fourhours, and two weeks following treatment of ES-2 xenograft mice with atartrate salt of a compound of structure (I). FIG. 75 shows the imagingresults of a Proteome Profiler Human Chemokine Array Kit (left panel)and the relative quantification of the cytokines/chemokines, as comparedto a vehicle control. As can be seen in FIG. 75, CXCF12 and G-CSF weredecreased following treatment with a tartrate salt of the compound ofstructure (I).

Example 33: EMT Markers in Ascites Fluid Following Treatment with aCompound of Structure (I)

FIG. 76 shows the mRNA expression levels of EMT markers assayed fromascites fluid following treatment with a tartrate salt of the compoundof structure (I). As can be seen in FIG. 76, EMT markers were notsuppressed in ascites fluid following treatment. In fact, GAS6, forexample, was increased as soon as two hours following treatment with atartrate salt of the compound of structure (I).

Example 34: Soluble Factors AXL, GAS6 and PD-L1 are Suppressed FollowingIn Vivo Treatment with a Compound of Structure (I)

FIG. 77 shows AXL, GAS6, and PD-L1 protein levels following treatmentwith either 25 mg/kg or 50 mg/kg of a tartrate salt of the compound ofstructure (I). Protein levels were measured by ELISA assay.

As can be seen in FIG. 77, AXL, GAS6, and PD-L1 were suppressedfollowing treatment with a tartrate salt of the compound of structure(I). To determine if the suppressed levels of AXL, GAS6, and PD-L1 weredue to suppression of these factors from the tumor cells, expression ofmouse versus human level of each factor was measured. As can be seen inFIG. 78, AXL was mainly released from the ES-2 cells (human cells),whereas GAS6 was mainly released from non-cancer cells (murine cells).PD-L1 was equivalently released from ES-2 cells and non-cancer cells.

Example 35: In Vivo Efficacy of the Compound of Structure (I) at a Doseof 60 mg/kg in a Mouse Ovarian Cancer Xenograft Model

In vivo efficacy was tested for 60 mg/kg of a tartrate salt of thecompound of structure (I) in intraperitoneal transplantation model ofES-2 ovarian clear cell carcinoma cell line. Vascular permeability dueto metastatic colonization of intraperitoneal membranes by tumor cellsis a major cause of ascites development in ovarian cancer patients. Thisxenograft model allowed for evaluation of the development of ascites,and peritoneal and mesenterial spread of tumor cells.

Ovarian clear cell carcinoma ES-2 cells were transfected with luciferaseexpression vector pGL4.1 (1×10{circumflex over ( )}⁶ cells) wereinjected intraperitoneally in to the BALB/cAnNCrj-nu/nu mice. After 5days post-injection, luciferase expression was measured, which indicatedcancer cells were growing intraperitoneally. Subsequently, 12 mice wererandomized into two groups for vehicle and 60 mg/kg of a tartrate saltof the compound of structure (I). The compound of structure (I) wasprepared in a vehicle of TPGS buffer and given by oral gavage. The drugswere provided a ‘5-day on, 2-day off’ schedule for 2 cycles. Representimages of mice were taken at day 14 (FIG. 79, top left panel). For micein both treatment groups (60 mg/kg of a tartrate salt of a compound ofstructure (I), versus a vehicle control), body weight is shown in FIG.79, top right panel; abdominal circumference is shown in FIG. 79 bottomleft panel; volume of recovered ascites is shown in FIG. 79, bottommiddle panel; and percent survival is shown in FIG. 79, bottom rightpanel (which additionally includes mice treated with cisplatin, “CDDP”,at 2.5 mg/kg, iv, 1/wk). Treatment with a tartrate salt of the compoundof structure (I) (60 mg/kg, PO, qd) significantly suppressed ascitesdevelopment (ascites volume, vehicle 3.34 mL, a tartrate salt of acompound of structure (I)-0.28 mL, abdominal circumference, vehicle—6.48cm (day 0) to 7.96 cm (day 15), a tartrate salt of a compound ofStructure (I) 6.55 cm (day 0) to 6.82 cm (day 15), p<0.05) without anybody weight loss. The compound of structure (I) also prolonged overallsurvival significantly compared to vehicle or cisplatin, “CDDP.”

Example 36: In Vivo Efficacy of the Compound of Structure (I) at a Doseof 60 mg/kg in a Mouse Ovarian Cancer Xenograft Model

Twenty-four hours following injection with ES-2 cells as described inExample 10, tumor cells were collected from abdomen, then lysed andwestern blotted for tubulin, AXL total and phosphorylated at Y702 site(FIG. 80, left panel). The plot shows the relative AXL phosphorylationfor the treatment group as compare to the vehicle-treated group (FIG.80, right panel). As shown in FIG. 80, a tartrate salt of the compoundof structure (I) at a dose of 60 mg/kg reduced at 24 hours the ratio ofp-AXL (Y702)/AXL in the collected tumor cells from abdomen. Theseresults indicated that a tartrate salt of the compound of structure (I)directly blocked the growth of metastasized ovarian cancer cells.

Example 37: OVCAR3 Tumor Ascites Model Development

The OVCAR-3 model for ovarian cancer induced-ascites employed in thisstudy was adapted from the following references: Hamilton et al. 1984.Can Res.; Hu et al. 2002. Am J Pathol. In brief, mice were injected with1×10⁷ OVCAR3 cells/mouse. Once subcutaneous tumors had begun to developat the site of injection, the abdominal circumference was monitoredregularly. FIG. 81 shows the abdominal circumference (left panel) andbody weight (right panel) of tumor-bearing versus non-tumor bearingmice. FIG. 82 shows the ascetic volume and representative photographs ofmice treated in the study, with non-tumor bearing mice shown on the leftand tumor bearing mice shown on the right. With the intraperitonealinjection of the OVCAR3 cells, mice exhibited increased abdominalcircumference and increased ascitic volume.

Example 38: In Vitro Cytotoxicity and In Vivo Efficacy of the Compoundof Structure (I) in an Intraperitoneal Xenograft Model

In vitro cytotoxicity was tested in a proliferation assay of A2780ciscells. Cell viability was evaluated by CellTiter-Glo assay. The resultsof treating the cells with varying doses of a tartrate salt of thecompound of structure (I) three days after treatment are shown in FIG.83.

In vivo efficacy was tested for vehicle (n=8), 15 mg/kg (n=8) and 30mg/kg (n=8) of a tartrate salt of the compound of structure (I) and CDDP5 mg/kg (n=8) in intraperitoneal transplantation model of A2780cisovarian carcinoma cell line. Ovarian carcinoma A2780cis cells(1×10{circumflex over ( )}7/mouse) were injected intraperitoneally in tomice. Five days after implant, drugs except for CDDP were orallyadministered once a day and CDDP was administered intraperitoneally oncea week. FIG. 84 shows the circumference and FIG. 85 shows the bodyweightof the mice 5-25 days after implant. FIG. 86 shows representativephotographs of mice in the study at day 25 after treatment, a vehicletreated mouse is on the left and a mouse treated with 30 mg/kg of atartrate salt of the compound of structure (I) is on the right.

Tumors were lysed and mRNA and protein were extracted four hours afterthe final administration of a tartrate salt of the compound of structure(I). Gene expression levels in the tumors were analyzed by RT-qPCR(n=7-8). The expression of CYP26A1, Snail, Slug, Twist, Axl, GAS6, CDH1,CDH2, and ZEB1, which are normalized by that of GAPDH, are shown in FIG.87.

As shown in FIG. 88, protein expression levels were measured by westernblot analysis for Snail and β-actin as a loading control followingtreatment with a tartrate salt of the compound of structure (I). Sampleswere pooled per each group (n=7-8).

Serum were collected four hours after the final administration of atartrate salt of the compound of structure (I). Human Axl expression inserum is measured by ELISA as shown in FIG. 89.

Example 39: Testing the Efficacy of a Compound of Structure (I) in anOVCAR3 Tumor Ascites Model

Following intraperitoneal inoculation with the OVCAR3 cells, mice aretreated with a tartrate salt of a compound of structure (I) (e.g., n=6)or a vehicle control (e.g., n=6), and the abdominal circumference andbody weight of each mouse is measured to determine the efficacy of atartrate salt of the compound of structure (I). Soluble AXL, GAS6, andPD-L1 levels are also measured from the mice in both treatment groups.

Example 40: Synthesis of Compound 1

Compound 1 is synthesized by reacting compound 1-A with 1-B using theconditions indicated above. Compound 1-C is then reacted with compound1-D using the reaction conditions shown to afford the product 1-E. Theamine protecting group is then removed using suitable acidic conditions(e.g., TFA or HCl in dioxane). Purification using standard techniques(e.g., silica gel chromatography or prep-HPLC) as needed.

Example 41: Synthesis of Compound 2

Compound 2 is synthesized by reacting compound 2-A with 2-B using theconditions indicated above. Compound 2-C is then reacted with compound2-D using the reaction conditions shown to afford the desired product(Compound 2). Purification using standard techniques (e.g., silica gelchromatography or prep-HPLC) as needed.

Example 42: Synthesis of Compound 3

Compound 3 is synthesized by reacting compound 3-A with 3-B using theconditions indicated above. Compound 3-C is then reacted with compound3-D using the reaction conditions shown to afford the product 3-E. Theamine protecting group is then removed using suitable acidic conditions(e.g., TFA or HCl in dioxane) to afford Compound 3. Purification usingstandard techniques (e.g., silica gel chromatography or prep-HPLC) asneeded.

Example 43: Synthesis of Compound 5

Compound 4-A is treated with Oxone® (i.e., potassium peroxymonosulfate)and deprotected using acidic conditions (e.g., TFA). The deprotectedproduct 4-B is then coupled with aldehyde 4-C via a reductive aminationto afford the desired product 4-D. The compound 4-D is then deprotectedto afford compound 4-1 as indicated above.

In parallel, compound 4-F is reacted with 4-G using the conditionsindicated above. Compound 4-H is then reacted with compound 4-1 preparedas described above to afford the desired product, Compound 5.Purification using standard techniques (e.g., silica gel chromatographyor prep-HPLC) as needed.

Example 44: Compound IV Metabolite PK Data

Representative exampled from subjects dosed with a compound of structure(IV) is presented in FIGS. 90-92 (16 mg/m²). The data shows theconcentration of the compound of structure (VI) and two metabolitescompounds (i.e., Compounds 2 and 3). Data was collected on day one ofthe dosing (D1) and on day 21 (D21).

As shown in FIG. 90, Compound 2 and Compound 3 were observed at relativeconcentration levels of approximately 1:1 and 1:10 with the compound ofstructure (IV), respectively, in this subject. As shown in FIG. 91,Compound 2 and Compound 3 were observed at relative concentration levelsof approximately 1:2 and 1:4 with the compound of structure (IV),respectively, in this subject. As shown in FIG. 92, Compound 2 andCompound 3 were observed at relative concentration levels ofapproximately 1:1 and 1:3 with the compound of structure (IV),respectively, in this subject.

Additionally, the overall pharmacokinetic profile for Compound 2 andCompound 3 parallels the compound of structure (VI) in terms of theT_(max) accumulation and clearance.

The mean area under the curve (AUC) from the initial time point untilthe last time point is shown in FIG. 93. The AUC values with metabolites(Compounds 2 and 3) are 2-3× higher than Compound VI alone. By day 21,the AUC values are proportionally higher (approximately 4-fold higher).

Example 46: Cardiovascular Safety Assessment of Compound of Structure(I) in Dogs

This example reports the study of the potential toxicity andtoxicokinetics of a tartrate salt of the compound of structure (I) whenadministered orally to dogs for 28 consecutive days as well as theprogression or regression of any effects following a 14-daytreatment-free recovery period.

Electrocardiograms (6 limb leads) were obtained for all animals onceduring the pre-treatment period and on Day 27 of dosing at approximately1.5 hours post dosing; close to the Tmax (2 hr±30 minutes). Tracingswere assessed for gross changes indicative of cardiac electricalabnormalities. Heart rate (HR) (lead II), rhythm or conductionabnormalities were also evaluated. Each ECG was evaluated for HR,rhythm, P wave duration, QRS duration, PR interval, and QT duration. Themorphology of the QRS complexes was evaluated in the frontal plane leadsfor gross abnormalities. Electrocardiograms were evaluated by aBoard-certified Veterinary Cardiologist.

Blood pressure (BP) using a non-invasive technique was recorded atapproximately the same time as ECGs, and included HR, andsystolic/diastolic BP.

The heart rate, PR interval, P wave duration and QRS duration werewithin normal limits for all groups and there was no significantdifference noted between groups at the different time points. The QTinterval was within normal limits for all groups and all time points.Pre-study HR was significantly lower in the high dose group compared toall other groups and was considered an incidental finding. Noarrhythmias were noted in the different groups at the different timepoints.

Example 47: Additional Pre-Clinical Studies; Safety Pharmacology andAnimal Toxicity/Pharmacokinetics of Compound of Structure (I)

In Vivo Pharmacodynamic and Biomarker Studies

In order to determine bio marker movement in vivo, CYP26A1 levels weremeasured in tumors from mice treated with a tartrate salt of a compoundof structure (I). Tumor tissues from athymic nude mice bearingsubcutaneously xenografted MV4-11 tumors were removed at 6 or 24 hoursfollowing a single oral dose of the compound (180 mg/kg). CYP26A1 levelswere measured by immunohistochemistry (IHC) as well as by RT-qPCRassessment. Results showed that a compound of structure (I) attenuatesAXL-associated biomarkers in vivo.

Safety Pharmacology

A number of assays were performed to evaluate the potential effects of atartrate salt of the compound of structure (I) on hERG activity.

(i) Effects on hERG Channel Function

Biochemical binding to the hERG channel was evaluated using thePredictor™ hERG Fluorescence Polarization Assay (Invitrogen). Resultsshowed that a tartrate salt of a compound of structure (I) is a weakbiochemical inhibitor of hERG activity, exhibiting an IC₅₀ greater than1 μM. In addition, 3 separate hERG patch-clamp experiments were alsoconducted, 2 using HEK-293 cells and 1 using CHO cells. The data fromcell-based Study No. 191081 suggests that a tartrate salt of thecompound of structure (I) inhibits hERG activity by greater than 50% at1 μM in transfected HEK-293 cells. However, data from the 2 otherpatch-clamp assays conducted in both HEK-293 and CHO cells are moreconsistent with the data from the cell-free biochemical screen,concluding that hERG inhibition is not significant (>50%) below 1 μM ofa tartrate salt of the compound of structure (I).

(ii) Receptor Panel Screening

The tartrate salt of the compound of structure (I) was screened throughthe “Safety 44 panel” of safety-associated receptors and enzymes offeredby Cerep. The compound of structure (I) inhibited only 15 of theseassays when screened at the highest dose tested at 10 μM. Based onanimal pharmacokinetic data, animal efficacy models and toxicology data,10 μM concentrations have not been reached in animals and are notexpected to be achieved in future human studies. Therefore, off-targetreceptor inhibition is not expected to lead to significant toxicityconcerns in future testing of the compound of structure (I).

(iii) Cardiovascular Safety Assessment in Dogs

A study was carried out to examine the potential toxicity andtoxicokinetics of a tartrate salt of the compound of structure (I) whenadministered orally to dogs for 28 consecutive days as well as theprogression or regression of any effects following a 14-daytreatment-free recovery period.

Electrocardiograms (ECGs) (6 limb leads) were obtained for all animalsonce during the pretreatment period and on Day 27 of dosing atapproximately 1.5 hours postdose; close to the time to C_(max) (peaktime) (T_(max)) (2 hr±30 minutes). Tracings were assessed for grosschanges indicative of cardiac electrical abnormalities. Heart rate (HR)(lead II), rhythm or conduction abnormalities were also evaluated. EachECG was evaluated for HR, rhythm, P wave duration, QRS duration, PRinterval, and QT duration. The morphology of the QRS complexes wasevaluated in the frontal plane leads for gross abnormalities. ECGs wereevaluated by a Board-certified Veterinary Cardiologist.

Blood pressure (BP) using a noninvasive technique was recorded atapproximately the same time as ECGs, and included HR, andsystolic/diastolic BP.

The HR, PR interval, P wave duration, and QRS duration were withinnormal limits for all groups and there was no significant differencenoted between groups at the different time points. The QT interval waswithin normal limits for all groups and all time points. Prestudy HR wassignificantly lower in the high dose group compared to all other groupsand was considered an incidental finding. No arrhythmias were noted inthe different groups at the different time points.

Nonclinical Absorption, Distribution, Metabolism and Excretion Studies

(i) Drug Transporter Effects

The tartrate salt of the compound of structure (I) was tested in abidirectional cell permeability assay using confluent monolayer ofCaco-2 cells in a 96-well based format. Fenoterol, Propranalol, andDigoxin were used as controls. The efflux ratio (mean apparentpermeability [Papp] A to B/mean Papp B to A) for a tartrate salt of thecompound of structure (I) was determined to be 1.49. Although massrecovery was low, this data suggests that the compound of structure (I)is not a substrate for P-glycoprotein and is a compound of moderatepermeability.

(ii) CYP450 Effects

A tartrate salt of the compound of structure (I) was evaluated for theinhibition of human cytochrome P450 (CYP) isozymes using human livermicrosomes in the presence of NADPH. At 10 μM, a tartrate salt of thecompound of structure (I) inhibited the activity of only isoform 2C19 bymore than 50% out of the isozymes selected for testing. The IC₅₀ valueswere determined against all the CYP isozymes used in the panel and,consistent with the percent inhibition data, only 2C19 was inhibited ata concentration lower than 10 μM (IC₅₀ 4.4 μM).

(iii) Liver Microsome Stability Studies

The stability of a tartrate salt of the compound of structure (I) in thepresence of isolated microsomes from 3 species (human, rat and dog) weredetermined. The concentration of a tartrate salt of the compound ofstructure (I) was measured by liquid chromatography-tandem massspectrometry (LC-MS/MS) with reference to a standard curve. The t_(1/2)of a tartrate salt of a compound of structure (I) ranged from 4.3minutes in humans to 4.9 minutes in dogs.

(iv) Serum Albumin Binding

The serum albumin binding levels for the compound of a tartrate salt ofcompound of structure (I) were determined using human plasma in adialysis plate-based assay. The free fraction of a tartrate salt of thecompound of structure (I) was measured by LC-MS/MS, by reference to astandard curve, and warfarin was used as a control. Data showed that thecompound of structure (I) has moderate human serum albumin binding (7%unbound). Recovery of protein bound drug (80.3%) indicated the bindingwas reversible.

(v) pKa of the Compound of Structure (I)

The pKa values for a tartrate salt of the compound of structure (I) weredetermined by titration using ultraviolet metric detection. The pKa wasdetermined in aqueous buffer and separately in the presence of 2cosolvents (80% methanol and 60% DMSO). The final pKa values werecalculated as an average of the 3 values obtained under differentsolvent conditions with the exception of pKa 3, for which the pKa valuedetermined in DMSO was excluded. The final pKa values for pKa1, pKa2,and pKa3 were 3.02, 3.96, and 7.81, respectively.

(vi) pH-Dependent Solubility of the Compound of Structure (I)

The equilibrium solubility of the compound of structure (I) tartrate wasdetermined in the following media: pH 3.5, 4.5, 5.5, 6.5 USP buffers,0.1N HCl, SGF, fasted state simulated intestinal fluid, and fed statesimulated intestinal fluid. As expected, based on the pKa determination,a tartrate salt of the compound of structure (I) showed greatestsolubility in acidic media.

Animal Toxicology and Pharmacokinetics

(i) 7-Day Repeated Oral Dose Range-Finding Study of the Compound ofStructure (I) with Single and Repeated Dose PK in Rats

A 7-day repeated oral dose range-finding study of a tartrate salt of thecompound of structure (I) with single and repeated dose pharmacokinetics(PK) was performed in rats. Groups of 4 male and 4 female rats received2, 4, or 8 mg/kg of a tartrate salt of the compound of structure (I)dissolved in 1% Tween 80/5% D-α-tocopherol polyethylene glycol 1000succinate (TPGS)/water (v/v/v) by oral gavage once daily for 7consecutive days followed by a 7-day observation period.

Analysis of all generated data, clinical observations, body weight, bodyweight changes, food consumption, clinical pathology evaluations, grossnecropsy, and organ weights revealed that dose levels of a tartrate saltof the compound of structure (I) at 2 and 4 mg/kg/day administeredorally once a day for 7 days were well tolerated in both males andfemale rats. The plasma levels and exposure (based on area under theplasma concentration curve [AUC] from time 0 to 12 hours (AUC₀₋₁₂)displayed a linear increase with dose following a single, 3 and 7 daysof dosing at 2 and 4 mg/kg/day with higher exposures in females comparedto males and plasma levels that did not change during treatment.

At a dose of 8 mg/kg, male rats tolerated the 7 day repeated doses of atartrate salt of the compound of structure (I) well and plasmaconcentrations of a tartrate salt of the compound of structure (I) didnot change during treatment. In contrast, female rats treated orallywith a tartrate salt of the compound of structure (I) at 8 mg/kg/dayshowed treatment-related toxicity including decreased food consumption,weight loss, pallor, diarrhea, and melena. The most prominenthematological finding at the end of treatment was a reduction of whiteblood cells associated with severe neutropenia. The main findings ingross necropsy for the female rats that were moribund sacrificed on Day8 were spleen enlargement, renal pallor, and bone marrow as well asreduction of abdominal fat and muscle mass (cachexia). Plasmaconcentrations of a tartrate salt of the compound of structure (I) infemale rats at a dose of 8 mg/kg were higher compared to male rats andmuch higher on Day 7 compared to Day 3 indicative of a dose accumulationeffect.

Pharmacokinetic studies of a tartrate salt of the compound of structure(I) revealed a rapid absorption with Tmax values similar, between maleand female rats, across all doses and days of dosing, ranging from0.5-2.0 hrs. The magnitude of the plasma AUC from time 0 to the time ofthe last concentration (AUC0-Tlast) displayed a gender bias, beingapproximately 1.5-2-fold higher in females compared to males, with theonly exception being high dose females following 7 days of dosing wherethe AUC0-Tlast was 5.8-fold higher. Maximum observed plasmaconcentration (Cmax) and AUC0-Tlast dropped slightly with increasingdays of dosing, with the exception of high dose females where AUC0-Tlastdid not change from Day 1 to Day 3 and increased from Day 3 to Day 7 byapproximately 2-fold. Following a single dose and 3 days of dosing theAUC0-12 increased in a linear fashion between doses for both male andfemale rats. On the seventh day of dosing, the relationship betweenincreasing dose and AUC0-12 deviated from linearity in female rats, dueto a larger than expected increase of the high dose AUC0-12 suggestingthat there was dose accumulation of the compound of structure (I).

(ii) 28-Day Repeated Oral Dose Toxicity and Toxicokinetic Study of theCompound of Structure (I) in Rats

The potential toxicity and toxicokinetics of a tartrate salt of thecompound of structure (I) when administered orally to rats daily for 28days was evaluated. Groups of 10 male and 10 female rats received 0.5,2, or 4 mg/kg/day of a tartrate salt of a compound of structure (I)dissolved in 1% Tween 80/5% TPGS/water (v/v/v) by oral gavage once dailyfor 28 consecutive days. A control group (10 males/10 females) was dosedwith the vehicle only. The progression or regression of any effects wereevaluated during an additional 14-day treatment-free period in thecontrol group and in rats dosed at 2 and 4 mg/kg/day.

There were no treatment-related observations recorded during the 28 daysof dosing or 14-day treatment-free period in the control group. Meanbody weights and mean body weight gains of the male rats in the highdose group were significantly lower at the end of treatment; however,this was not observed in the females of this group.

The main finding of toxicological relevance in the hematologyevaluations was a slight reduction of reticulocyte counts in the highdose group which may reflect reduced erythropoiesis. This findingappeared to be transient since reticulocyte counts in the high doserecovery group were comparable to the control group. Coagulation, serumchemistry, and urinalysis evaluations did not reveal any test itemtreatment-related findings.

The compound of structure (I) was rapidly absorbed following oraladministration, with the majority of the T_(max) values between 1-2 hrsin both males and females. There was no evidence of dose accumulationobserved on Days 1, 15, and 28 of dosing. Females had higher C_(max)values than males at all doses and on all days of dosing but there wereno differences in the T_(max) values and the higher C_(max) values infemales were not associated with differences in clinical, clinicalpathology and anatomo-histopathological findings. The eliminationhalf-life (t_(1/2(e))), determined from the available data of dosegroups of 2 and 4 mg/kg ranged from 2.3-8.9 hours and was similar inboth genders. The compound of structure (I) exhibited a high volume ofdistribution suggesting for a large tissue distribution.

There were no gross findings of toxicological relevance observed ingross necropsy examinations performed at the end of treatment andrecovery periods. The only finding of possible toxicological relevancein the organ weight evaluation was a lower weight of thyroid withparathyroids in male rats dosed with the test item; however, there wereno abnormal findings in the histopathology of thyroid and parathyroids.

The abnormalities found in histopathology of the high dose rats includedsmall acute hemorrhages in mesenteric lymph nodes in 5 animals and focalinflammatory infiltrates and/or focal lobular atrophy in pancreas of 3control and 7 high dose animals. These findings were equivocal and itcould not be determined whether they were related to test itemtreatment. While exposure to the test item had a dose-related effect onthe body weight of male rats in the high dose group and was associatedwith a transient mild reduction in erythropoiesis in males and females,no underlying mechanism of action was apparent from the histopathologyevaluations. Hematopoiesis evaluated in the spleen and bone marrowtissues were similar in the high dose and control groups.

Based on all data generated, including clinical observations, bodyweights, food consumption, ophthalmoscopy, clinical pathology,toxicokinetics, gross pathology and histopathology, the No ObservedAdverse Effect Level (NOAEL) of a tartrate salt of the compound ofstructure (I) following 28 days of repeated oral dosing in rats wasdetermined to be 2 mg/kg/day.

(iii) Single Oral Dose and 7-Day Repeated Oral Dose Toxicity andPharmacokinetic Study of the Compound of Structure (I) in Dogs

This study established a dose level by a single oral dose (Part A) anddetermined the toxicity and PK of a tartrate salt of the compound ofstructure (I) (Part B) dissolved in 1% Tween 80 and 5% vitamin-E TPGS inwater following 7 days of repeated oral dosing in Beagle dogs. Doseswere administered via a stomach tube at a dose volume of 2 mL/kg afterovernight fasting. Food was offered approximately 1 hour after dosing.

Three groups of dogs, each consisting of 1 male and 1 female, received asingle compound of structure (I) (tartrate) dose of 0.25, 0.5, or 1mg/kg and observed for 14 days (Part A). Salivation was observed in thehigh dose (1 mg/kg) male dog approximately 30 minutes postdose. Emesiswas observed in the high dose female dog approximately 60 minutespostdose. All dogs completed the treatment period and survived thescheduled observation period. There were no other observations oftoxicological relevance seen in hematology and serum chemistryevaluations.

In Part B of the study, 3 groups of dogs, each consisting of 2 males and2 females, received a tartrate salt of the compound of structure (I) for7 consecutive days at doses of 0.25, 0.75, or 1.25 mg/kg/day followed by7-day observation period. A control group comprised of 1 male and 1female received vehicle only.

All dogs completed the treatment and observation periods and survived toscheduled necropsy. Analysis of all generated data, including clinicalobservations, body weights, food consumption, clinical pathology, grossnecropsy, and organ weights revealed no test article treatment-relatedsignificant toxicity in dogs that were treated orally with a tartratesalt of the compound of structure (I) up to 1.25 mg/kg/day for 7 days.Clinically, a tartrate salt of a compound of structure (I) caused emesisat the initial dose level of 0.25 mg/kg/day and up to 1.25 mg/kg/day.

Pharmacokinetic studies of a tartrate salt of the compound of structure(I) revealed a rapid absorption with T_(max) values similar between maleand female dogs, across all doses and days of dosing, ranging from0.5-6.0 hours. The magnitude of the plasma AUC_(0-Tlast) did not displaya gender bias. Oral t_((1/2)e) and mean residence time (MRT) values wereslightly longer following 7 days of dosing. The AUC_(0-Tlast) increasedlinearly with dose across genders and following a single and 7 days ofdosing. At the doses employed, there was little evidence of doseaccumulation for the compound of structure (I) in Beagle dogs following7 days of repeated dosing.

(iv) Seven-Day, Three Times Per Day, Repeated Oral Dose Toxicity andPharmacokinetic Study of the Compound of Structure (I) in Dogs

This study evaluated the toxicity and pharmacokinetics of a tartratesalt of the compound of structure (I) following 7 days of 3 times a day,repeated oral dosing in dogs. This study was performed following the7-day repeat dose, once a day repeat dose study to support thedetermination of the MTD of a tartrate salt of the compound of structure(I) in dogs.

To achieve sufficient systemic exposure at the high dose levels, 2groups of 2 females were dosed with a tartrate salt of the compound ofstructure (I) incorporated in gelatin capsules at dose levels of 3 or 6mg/kg/day (1 or 2 mg/kg/dose).

In the 3 mg/kg/day dose group, both dogs had emesis on most of thedosing days with the vomiting time ranging from approximately 15 minutesto 2 or 6 hours postdose. Soft feces or diarrhea was observed from Days3 or 7 to 14. One of the dogs had a 1.1 kg body weight loss over the7-day treatment period and started to regain body weight, gaining 400 g,during the 7-day recovery period. The other dog gained 300 and 600 gramsbody weight over the 7 and 14-day periods, respectively.

Both dogs in the 6 mg/kg/day (2 mg/kg/dose×3 days) dose group, showedvarious signs of toxicity early in the treatment phase. The severity ofthe clinical signs increased, and the health condition of the dogsdeteriorated after each subsequent dose. The clinical signs mostlyconsisted of vomition, diarrhea, emaciation, anorexia, dehydration,apathy, and weight loss. Due to deteriorating health, the 2 dogs in thisgroup were dosed only twice a day on Day 4 and dosing was skipped onDays 5 and 6, but the dogs were dosed for the PK evaluation on Day 7.After the end of treatment, the condition of animals continued todeteriorate, and their condition did not improve despite support withfluid therapy provided on Day 9. One dog was found dead the morning ofDay 10 and the second dog was euthanized in moribund condition that sameday.

Clinical pathology results in the dogs dosed at 6 mg/kg/day, on Day 8,showed slight increases in white blood cells (WBCs), red blood cells(RBCs) and neutrophils; increased hematocrit and hemoglobin values; anddecreased reticulocyte counts in one of the dogs. In the other dog,there were increases in WBCs, RBCs, neutrophils, monocytes, andbasophils; increased hematocrit and hemoglobin values; and decreasedreticulocyte counts and platelets. In the dogs dosed at 3 mg/kg/day, onDay 8, all hematology parameters were within the normal ranges for ofthe dogs. In the second dog there was a slight increase in RBC count;increased hematocrit and hemoglobin values; and reticulocyte count,although within the normal range, was decreased compared to thepretreatment count.

On Day 14, hematology results were within the normal reference rangeswith exception of a slight increase in reticulocyte counts in 1 of thedogs of this group. The only serum chemistry parameters that wereaffected in the dogs dosed at 3 mg/kg/day were elevated ALT (almosttwice the normal upper limit) in 1 of the dogs and elevatedtriglycerides in both dogs.

Pharmacokinetic analysis revealed that the plasma exposure to a tartratesalt of the compound of structure (I) as measured by AUC from time 0 toinfinity (AUC_(0-inf)) was not dose proportional and higher on Day 7compared to Day 1 following the third dosing, and even more so for the 6mg/kg/day dose compared to the 3 mg/kg/day dose. These observations weremade despite the vomiting that occurred and a partial cessation ofdosing at the 6 mg/kg/day dose, suggesting that there may be a tendencyfor dose-accumulation following consecutive days of dosing. C_(max)values were minimally impacted while T_(m)ax values were longer at the 6mg/kg/day dose. Both the t_((1/2)e) and MRT values were slightly longerwith increased days of dosing suggesting a change in the clearancemechanisms for the compound of structure (I) with consecutive days ofdosing.

Necropsy was performed on the dogs that were unscheduled sacrificed andfound dead on Day 10. At necropsy, cachexia and dark yellowish andwatery lower intestinal contents were observed in 1 dog. In the otherdog, cachexia, hyperemia of the glandular portion of the stomach;hemorrhagic lower intestines and rectum; dark reddish black color oflower intestinal contents; and hyperemia in the cortex and medulla ofthe kidneys were observed.

The MTD of a tartrate salt of the compound of structure (I) was reachedfollowing dosing of 1 mg/kg/dose 3 times per day (3 mg/kg/day) and thedogs recovered well within 1 week of cessation of treatment.

(v) 28-Day Repeated Oral Dose Toxicity Study of the Compound ofStructure (I) in Beagle Dogs Followed by a 14-Day Recovery Period

This study examined the potential toxicity and toxicokinetics of atartrate salt of the compound of structure (I) when administered orallyto dogs for 28 consecutive days. The progression or regression of anyeffects following a 14-day treatment-free recovery period was alsoassessed. Two groups of dogs (3 males/3 females) were dosed with atartrate salt of the compound of structure (I) incorporated in gelatincapsules at the following dose levels: 0.1, 0.5 mg/kg/day for 28 daysand another group was dosed at 1 mg/kg/day for the first 14 days,followed by 2 mg/kg/day for the remaining 14 days of the treatmentperiod. A control group of dogs was included in the study and was dosedwith empty gelatin capsules.

All dogs completed the 28-day treatment period and survived untilscheduled termination for necropsy. Clinical signs noted in thehigh-dose group during the treatment period included vomition,salivation, diarrhea, and/or soft feces. Vomition affected all dogs inthe high-dose group and was observed in some dogs in the mid-dose group.Reduced food consumption was observed in 1 male and 1 female; whereasweight loss was observed in 1 out of 5 males and 2 out of 5 females inthe high-dose groups during Weeks 3 and/or 4 of treatment and followingthe dose increase from 1 mg/kg to 2 mg/kg. These signs were not observedat the end of the recovery period.

There were no ophthalmoscopy findings at the end of treatment in any ofthe animals. Evaluation of clinical pathology data (hematology,coagulation, serum chemistry, and urinalysis) did not reveal anyfindings clearly attributable to the test item. The compound ofstructure (I) resulted in a dose-dependent increase in plasmaconcentrations. The compound of structure (I) was quantified in someplasma samples following dosing at 0.1 mg/kg, however most plasmaconcentrations were below the lower limit of quantitation (0.2 ng/mL).The dose proportionality and linearity of plasma concentrations of thecompound of structure (I) following oral dosing could not be wellcharacterized given the low plasma levels at the lowest dose. However, acomparison of the mid and high doses suggests that plasma concentrationsin male and female dogs increased approximately in a dose proportionalmanner. At both the mid and high doses, all predose levels of thecompound of structure (I) were below the limit of quantification and forthe high dose, the elimination half-lives were similar on all days ofdosing, suggesting a lack of dose accumulation. The analysis oftoxicokinetic data revealed a high clearance of the compound ofstructure (I) in plasma that was caused from a large volume ofdistribution suggesting that a tartrate salt of the compound ofstructure (I) was well distributed in tissues.

Gross pathology findings observed in a few animals in the high-dosegroup included hemorrhage in colon of 3 animals and cecum of 1 animal,as well as hyperemia in the pylorus in 1 dog and hyperemia in the rectumin another dog. The hemorrhages were likely agonal, but might indicaterecent injury to the mucosa. No gross findings were observed in recoveryanimals in the high- and mid-dose groups.

There were no apparent differences in absolute and relative organweights between the control group and test item groups (both genders)with the exception of a few incidental significant differences: highermean weights of thyroids with parathyroids in the low-dose males and anincrease in the mean weights for uterus sizes in Group 4 females, due tophysiological luteal phase with endometrial hypertrophy.

Histopathological findings of possible toxicological significanceincluded changes in the thymus and the lower gastrointestinal tracttissues:

-   -   Thymic atrophy was found at the end of the treatment period in 3        out of 6 high-dose animals, 3 out of 6 mid-dose animals, 1 out        of 6 low-dose animals, and 1 out of 6 control animals. This        finding was also noted in 2 of 4 dogs in both the high- and        mid-dose groups at the end of the recovery period. Thymic        atrophy is a physiological response to stress and an expected        finding in young dogs around the onset of sexual maturity, so        these findings are considered to be nonspecific and indirectly        related to the treatment-associated gastrointestinal effects.    -   Minimal or mild degree of mucosal injury and inflammation in the        small intestine were observed at the end of the treatment period        in 1 mid-dose dog, 4 high-dose dogs, and 1 dog in the control        group. At the end of the recovery period, 1 control dog, 1        mid-dose dog, and 2 high-dose dogs had similar mild residual        changes. The small intestinal mucosal injury associated with the        high-dose regimen was considered to have returned to background        level at the end of the 14-day recovery period.    -   At the end of the treatment period, minimal or mild degree of        mucosal injury and inflammation in the colonic mucosa was        observed histologically in four animals in the high-dose group.        These responses were characterized by increased amounts of cell        debris in a few glands, associated with patchy neutrophil        infiltrates, or focal areas of hemorrhage in the lamina propria.        One control animal also had minimal injury to colonic glands        without inflammation or hemorrhage. Similar findings were not        observed in the large intestines in any of the animals of the        mid-dose group. Minimal degeneration of the colon mucosal glands        was observed at the end of the recovery period in one high-dose        dog.

Evaluation of clinical observations, body weight assessment, foodconsumption, ophthalmology, ECGs, clinical pathology, gross necropsy,and organ weights did not reveal any findings of clinical ortoxicological relevance in dogs dosed at 0.1 mg/kg/day. With theexception of vomiting, no other adverse findings of toxicologicalsignificance were found in dogs dosed with a tartrate salt of thecompound of structure (I) at 0.5 mg/kg/day. Emetogenic effect is acommon finding observed in dogs dosed with kinase inhibitor drugs andcould be attributed in part to a higher susceptibility of this speciestoward this class of drugs.

Administration of a tartrate salt of a compound of structure (I) at adose of 1 mg/kg/day for 14 days, followed by 2 mg/kg/day for 14 dayswas, for the most part, well tolerated by young male and female Beagledogs used in this study. Emesis was the most obvious adverse effectoccurring in this dose group and was more pronounced upon the doseincrease to 2 mg/kg. Other clinical signs, including salivation,diarrhea or soft feces and reduction of body weight gains, were observedduring the third and/or fourth weeks of treatment. Upon the ceasing oftreatment, there were no clinical signs observed in the recoveryanimals. Histopathological findings indicated that the potential targetorgans of toxicity were the lower gastrointestinal tract and thymus.Upon completion of the treatment period, there were no clinical signs orhistopathological findings observed in the recovery animals with theexception of thymic atrophy which was considered to be a stress-relatednonspecific response and may have occurred during the treatment phase.

Example 48: A Phase 1a/1b Dose-Escalation, Safety, Pharmacodynamic Studyin Patients with Advanced Solid Tumor

This study enrolls a sufficient number of patients to establish themaximum tolerated dose (MTD) of a tartrate salt of a compound ofstructure (I) (approximately 40 patients) in the Phase 1a part of thestudy, and then up to 100 additional patients in the Phase 1b part(Expansion at the MTD and Biopsy Cohorts). Enrolled patients receive thestudy drug disclosed herein administered once daily for the first 21 outof 28 days. Patients who successfully complete a 4-week treatment cyclewithout evidence of significant treatment-related toxicity orprogressive disease continue to receive treatment with the same dose anddosing schedule.

This study determines MTD and DLTs of oral, daily administration of thestudy drug for the first 21 days every 4 weeks, over a range of doses inpatients with advanced solid tumors, establishing pharmacokinetics ofthe oral administration, and/or observing patients for antitumoractivities of the study drug by objective radiographic assessment. Forexample, pharmacodynamics of the therapy include assessing biomarkers intumor tissues, in peripheral blood mononuclear cells (PBMCs), plasma,and serum and determining in vivo markers of AXL deregulation (inpatients treated at the MTD) by: evaluating tumor biopsies in patientswith easily accessible, low-risk tumors (as defined by localinterventional radiology), and/or assessing immune function and/orresponse using immunohistochemistry (IHC), flow cytometry, or othermolecular methodologies.

Study Drug

The compound of structure (I) is a novel oral inhibitor that targets AXLkinase and reverses the mesenchymal phenotype associated with advancedcancers. Preclinical studies have shown promising antitumor activity ofcompound of structure (I) as a single agent against a variety of tumortypes in both in vitro and in vivo studies. This first-in-human Phase1a/1b study with the compound of structure (I) is conducted in patientswith refractory solid tumors. The Phase 1a study is designed to identifythe maximum tolerated dose (MTD) and to identify the safety profile andRecommended Phase 2 Dose (RP2D) of compound of structure (I). Once theMTD has been established, 5 additional cohorts of up to 20 patients eachwith specific tumor types (up to 100 additional patients total) areenrolled at the MTD in the Phase 1b study. Data collected from patientsenrolled in each of these additional cohorts is used to confirm safety,explore potential biomarkers, and evaluate potential signals of activitywhen compound of structure (I) is administered to specific groups ofheavily pretreated patients or given in combination with immunotherapyor a tyrosine kinase inhibitor (TKI). Compound of structure (I) isadministered once a day, orally, for 21 days followed by a 7-day drugfree period. In some instances, the 28-day cycle is repeated if thepatient continues to show benefit and if compound of structure (I) isreasonably well tolerated.

Two drug substances are investigated in this clinical studies, amono-tartrate salt of the compound of structure (I) and a di-tartratesalt of the compound of structure (I) (Form A; “study drug”). Resultsfrom additional development work on the compound of structure (I) drugsubstance including testing multiple tartrate salt polymorph forms forsolubility, stability, reproducibility upon preparation and otherphysical attributes, as well as toxicology and pharmacokinetics (seeExamples 23-27), the di-tartrate form of the drug substance, Form A, hasbeen selected for further clinical use (“study drug”). The polymorphforms tested all exhibited similar solubility, toxicity, andpharmacokinetics characteristics compared to the drug substance used inthe former clinical form of the compound of structure (I) (Form B). Thedi-tartrate salt form of the compound of structure (I) that was selectedwas based on reproducibility compared to the current clinical form.

A summary of important chemical characteristics of both drug substanceforms is presented in Table 18.

TABLE 18 Nomenclature and Notable Chemical Characteristics of ClinicalDrug Substance Forms (Form B and Form A) Clinical Drug SubstanceChemical Clinical Drug Substance (Form A) (“study drug”) Characteristic(Form B) (free base:tartaric acid ratio of 1:2) Chemical{[5-chloro-2-([{4-methylpiperazin-1-{[5-chloro-2-({4-[{4-methylpiperazin-1- Name yl)methyl]phenyl}amino)pyrimidin-4- yl)methyl]phenyl} amino)pyrimidin-4- (IUPAC)yl]amino}-N,N-dimethylbenzene-1- yl]amino}-N,N-dimethylbenzene-1-sulfonamide mono-tartrate salt sulfonamide di-(L)-tartrate saltDescription White to off-white powder Off-white to yellow- tobrown-colored powder Chemical C₂₈H₃₆ClN₇O₈S C₃₂H₄₂ClN₇O₁₄S FormulaMolecular 666.15 Daltons 816.23 Daltons Weight Structural Formula

 

 

The study drug is supplied in oral form as a powder in hard gelatincapsules (size #3 for the 1-, 4-, 16-, and 25-mg doses; size #0 for the100-mg dose) and is manufactured under current Good ManufacturingPractices (cGMP) for investigational use.

Study drug capsules are formulated in 1-mg, 4-mg, 16-mg, 25-mg, and 100mg strengths and are packaged into round high-density polyethylenebottles with polyester coils as headspace fillers. Bottles are thenheat-sealed, fitted with child-resistant caps, and placed in low-densitypolyethylene bags as secondary packaging.

Clinical Results

Pharmacokinetic data from 9 patients treated at the first 3 dose levelsusing Form B of the compound of structure (I) demonstrated higherC_(max) and AUC of compound of structure (I) on Day 21 versus Day 1during Cycle 1, suggesting study drug accumulation. In addition,compound of structure (I) was detectable predose on Day 21 in 8 out of 9patients. In order to further characterize the predose levels ofcompound of structure (I), additional PK samples are collected predoseon Days 8 and 15 of Cycle 1; Days 1, 8, 15, and 21 of Cycle 2; and Day 1of Cycle 3.

Data has been collected to support the target level of the compound ofstructure (I). Currently, dosing is based on a standard mg/m² approach.Alternatively, flat dosing can be used. There may be less interpatientvariability in drug exposure when utilizing flat dosing versusBSA-dependent dosing. Flat dosing starts at the MTD. An average dose iscalculated based on the dose administered in the MTD expansion safetycohort.

Preclinical studies suggest that bone marrow, gastrointestinal tract,and the thymus may be potential target organs of toxicity. In the rat,reticulocyte counts were reduced; however, these findings were notobserved in the dog. In the dog, minimal to mild injury to the mucosallining as well as inflammation was noted in the small intestines andcolonic mucosa. Thymic atrophy was also noted in the dog. With theexception of vomiting, no other adverse effects of significance wereobserved in the dog. The preclinical GLP toxicology studies suggest thatthe observed toxicities were reversible following a recovery period of14 days with the exception of thymic atrophy.

Patients over 7 different dose cohorts (See, e.g., Table 19 below) hadbeen treated with a tartrate salt of compound of structure (I) in thisstudy (Form B). No dose-limiting toxicities (DLTs) had been reported.National Cancer Institute Common Terminology Criteria for Adverse Events(CTCAE) Grade 3 and 4 events that occurred in one patient each includedanemia, hypocalcemia, hypokalemia, hyponatremia, pleural effusion,urinary tract infection, ascites, hyperkalemia, hypertension, bacterialperitonitis, sciatica, and syncope. None of these events was consideredstudy drug-related. The most common Grade 1 and 2 AEs (ie, those thatoccurred in ≥3 patients each [13%]) included nausea, vomiting, fatigue,anemia, diarrhea, hypoalbuminemia, decreased appetite, hypomagnesemia,tachycardia, and thrombocytopenia. Grade 1 and 2 AEs that occurred in ≥3patients each (13%) and judged as at least Possibly related to thecompound of structure (I) included diarrhea, nausea, vomiting,dysgeusia, and thrombocytopenia. There were no serious AEs or deaths onstudy that were considered related to study drug.

TABLE 19 Patients on study with stable disease at least 4 cycles CohortDose Subject Histology # cycles Best Response 1 1.5 mg/m² 102 BreastCarcinoma (ER+/−HER2−) 8 SD 2 3 mg/m² 104 Neuroendocrine carcinoma 12 SD 3 6 mg/m² 108 Prostate adenocarcinoma 8 SD 4 9 mg/m² 109 Colonadenocarcinoma (KRAS mutant) 6 SD 4 9 mg/m² 110 Melanoma (BRAF wildtype)4 PR 4 9 mg/m² 203 Cholangiocarcinoma 4 SD 5 12 mg/m² 111 Uterinecarcinoma 6 SD 5 12 mg/m² 204 Pancreatic 8 SD 7 21 mg/m² 115 NSCLC 7(ongoing)

No dose-limiting toxicities (DLTs) had been reported. National CancerInstitute Common Terminology Criteria for Adverse Events (CTCAE) Grade 3and 4 events that occurred in one patient each included anemia,hypocalcemia, hypokalemia, hyponatremia, pleural effusion, urinary tractinfection, ascites, hyperkalemia, hypertension, bacterial peritonitis,sciatica, and syncope. None of these events was considered studydrug-related. The most common Grade 1 and 2 AEs (i.e., those thatoccurred in ≥3 patients each [13%]) included nausea, vomiting, fatigue,anemia, diarrhea, hypoalbuminemia, decreased appetite, hypomagnesemia,tachycardia, and thrombocytopenia. Grade 1 and 2 AEs that occurred in ≥3patients each (13%) and judged as at least possibly related to compoundof structure (I) included diarrhea, nausea, vomiting, dysgeusia, andthrombocytopenia. There were no serious AEs or deaths on study that wereconsidered related to study drug.

Study Design

This is a Phase 1a/1b, open-label, dose-escalation, safety,pharmacokinetics, and pharmacodynamic study.

(i) Phase 1a—Dose Escalation

The starting drug dose is 1.5 mg/m2 for 21 out of 28 days using astandard 3+3 design. Sequential cohorts of three (3) patients aretreated with escalated doses until the MTD is established. In theabsence of dose-limiting toxicities (DLTs), the dose is increased usinga modified Fibonacci dose escalation scheme.

If a patient experiences a DLT, up to three additional patients istreated at that dose level. If no additional DLTs are observed in theexpanded six-patient cohort, the dose is escalated in a new cohort ofthree patients. If two or more patients at a given dose level experiencea DLT during the first cycle, then the MTD have been exceeded and up toa total of six patients will be treated at the previous lower doselevel. If 0 or 1 of 6 patients experiences a DLT at this previous lowerdose level, this dose is declared the MTD.

(ii) Phase 1b—Expansion at MTD and Biopsy Cohorts

Once the MTD has been established, 5 additional cohorts of up to 20patients each with specific tumor types (up to 100 additional patientstotal) are enrolled at the MTD. Data collected from patients enrolled ineach of these additional cohorts is used to confirm safety, explorepotential biomarkers, and evaluate potential signals of activity whenthe drug is administered to specific groups of heavily pretreatedpatients or given in combination with immunotherapy or a tyrosine kinaseinhibitor (TKI). Ten patients in each of these 5 Expansion Cohorts arerequired to consent to undergo pre- and post-dose tumor biopsies. Allpatients who undergo these biopsies comprise the ‘Biopsy Cohorts’.

Patients may continue to receive study drug in 28-day cycles at the samedose given during Cycle 1 until they experience unacceptable toxicity orunequivocal disease progression. No intra-patient escalation of thestudy drug dose is permitted. Once the MTD has been established, dosingchanges from BSA-dependent to a flat dose based on the average of thedose administered in the MTD expansion safety cohort.

Patient Populations

(i) Phase 1a—Dose Escalation

Patients with histologically confirmed solid tumors who have showndisease progression after receiving standard/approved chemotherapy orfor whom there is no curative therapy.

(ii) Phase 1b—Expansion at MTD and Biopsy Cohorts

Given the fact that AXL is widely distributed in a variety of solidmalignancies, the dose expansion phase of this study at the MTD includesfive cohorts of specific patient tumor types to further assess thesafety and uncover possible signals of efficacy when the study drug isadministered as a single agent in groups of heavily pretreated patientsor in combination regimens in patients with immunotherapy-resistanttumors or EGFR⁺ NSCLC.

-   -   a. Patients with tumors that have progressed after achieving a        best documented response of at least stable disease (ie, SD, PR,        or CR documented per iRECIST) following at least 2 cycles (8        weeks) of immunotherapy and are felt to be appropriate for this        type of treatment.*    -   b. Patients with EGFR+ non-small cell lung cancer (NSCLC) who        have demonstrated recent progression following a best documented        response of at least stable disease (ie, SD, PR, or CR        documented per RECIST v1.1) on ≤2 lines of oral TKIs and are        felt to be appropriate for this type of treatment.* Prior        chemotherapy±immunotherapy is allowed as long as the patient is        clearly demonstrating current progression on an EGFR TKI. *        Patients with immunotherapy-resistant tumors or EGFR+ NSCLC who        are enrolled in these expansion cohorts continue treatment with        their previous immunotherapy or TKI regimens, respectively, and        add study drug. The rationale is based on preclinical data that        has shown that the combination is superior in patients who have        progressed on prior immunotherapy or a TKI.    -   c. Patients with BRAF-, KRAS-, or NRAS-mutated colorectal        carcinoma (CRC) for whom there is no standard therapy remaining.    -   d. Patients with persistent/recurrent ovarian cancer who would        be platinum refractory/resistant and have had any number of        lines of prior therapy,    -   e. Patients with BRAF-mutated melanoma who have progressed on        either immunotherapy or a combination BRAF/MEK inhibitor.        Inclusion Criteria:

To be eligible for participation in the study, patients must meet all ofthe following inclusion criteria:

1. Patients enrolled in the Phase 1a study must:

-   -   a. Have a histologically confirmed diagnosis of advanced        metastatic or progressive solid tumor.    -   b. Be refractory to, or intolerant of, established therapy known        to provide clinical benefit for their condition.

2. Patients enrolled in the Phase 1b study must meet criteria for one ofthe following tumor types:

-   -   a. Have tumors that have progressed after achieving a best        documented response of at least stable disease (ie, SD, PR, or        CR documented per iRECIST) following at least 2 cycles (8 weeks)        of immunotherapy and are felt to be appropriate for this type of        treatment.    -   b. Have EGFR+ NSCLC and have demonstrated recent progression        following a best documented response of at least stable disease        (ie, SD, PR, or CR documented per RECIST v1.1) on ≤2 lines of        oral TKIs and are felt to be appropriate for this type of        treatment. Prior chemotherapy±immunotherapy is allowed as long        as the patient is clearly demonstrating current progression on        an EGFR TKI.    -   c. Have BRAF-, KRAS-, or NRAS-mutated CRC for whom there is no        standard therapy remaining.    -   d. Have persistent/recurrent ovarian cancer who would be        platinum refractory/resistant and have had any number of lines        of prior therapy.    -   e. Have BRAF-mutated melanoma that has not responded to        immunotherapy or a combination BRAF/MEK inhibitor

3. Have one or more tumors measurable or evaluable as outlined bymodified RECIST v1.1 or iRECIST

4. Have an Eastern Cooperative Oncology Group (ECOG) (World HealthOrganization [WHO]) performance of ≤1

5. Have a life expectancy≥3 months

6. Be ≥18 years of age

7. Have a negative pregnancy test (if female of childbearing potential)

8. Have acceptable liver function:

-   -   a. Bilirubin≤1.5× upper limit of normal (ULN). Patients        receiving immunotherapy should have a bilirubin level≤3.0×ULN.    -   b. Aspartate aminotransferase (AST/SGOT), alanine        aminotransferase (ALT/SGPT) and alkaline phosphatase≤2.5× upper        limit of normal (ULN). If liver metastases are present, then        ≤5×ULN is allowed. Patients receiving immunotherapy should have        AST and ALT levels<5.0×ULN.

9. Have acceptable renal function:

-   -   a. Calculated creatinine clearance≥30 mL/min

10. Have acceptable hematologic status:

-   -   a. Granulocyte≥1500 cells/mm3    -   b. Platelet count≥100,000 (plt/mm3)    -   c. Hemoglobin≥9 g/dL

11. Have no clinically significant abnormalities on urinalysis

12. Have acceptable coagulation status:

-   -   a. Prothrombin time (PT) within 1.5× normal limits    -   b. Activated partial thromboplastin time (aPTT) within 1.5×        normal limits

13. Be nonfertile or agree to use an adequate method of contraception.Sexually active patients and their partners must use an effective methodof contraception (hormonal or barrier method of birth control; orabstinence) prior to study entry and for the duration of studyparticipation and for at least 30 days after the last study drug dose(see Section 4.6.3). Should a woman become pregnant or suspect she ispregnant while participating in this study, she should inform hertreating physician immediately.

14. Have read and signed the IRB-approved informed consent form prior toany study related procedure. (In the event that the patient isre-screened for study participation or a protocol amendment alters thecare of an ongoing patient, a new informed consent form must be signed.)

15. Patients enrolled in each of the five Expansion Cohorts must bewilling to consider pre-study and on-study biopsies, if safe andmedically feasible, as determined by local interventional radiology (3to 5 core samples requested at each biopsy timepoint).

Exclusion Criteria:

Patients meeting any one of these exclusion criteria will be prohibitedfrom participating in this study:

1. Have New York Heart Association (NYHA) Class III or IV, cardiacdisease, myocardial infarction within the past 6 months prior to Day 1,unstable arrhythmia, or evidence of ischemia on electrocardiogram (ECG)or during Cardiac Stress Testing within 14 days prior to Day 1.

2. Have a corrected QT interval (QTcF, Fridericia's method) of >450 msecin men and >470 msec in women

3. Have a seizure disorders requiring anticonvulsant therapy

4. Presence of symptomatic central nervous system metastatic disease ordisease that requires local therapy such as radiotherapy, surgery, orincreasing dose of steroids within 2 weeks prior to Day 1

5. Have severe chronic obstructive pulmonary disease with hypoxemia(defined as resting O2 saturation of ≤88% breathing room air)

6. Have undergone major surgery, other than diagnostic surgery, within 2weeks prior to Day 1

7. Have active, uncontrolled bacterial, viral, or fungal infections,requiring systemic therapy

8. Are pregnant or nursing

9. Received treatment with radiation therapy, surgery, chemotherapy, orinvestigational therapy within 28 days or 5 half lives, whichever occursfirst, prior to study entry (6 weeks for nitrosoureas or Mitomycin C)

-   -   a. This exclusion criterion is not applicable for patients with        EGFR+ NSCLC or immunotherapy-resistant tumors who are enrolled        in expansion cohorts at the MTD.

10. Are unwilling or unable to comply with procedures required in thisprotocol

11. Have known infection with human immunodeficiency virus (HIV),hepatitis B, or hepatitis C. Patients with history of chronic hepatitisthat is currently not active are eligible

12. Have a serious nonmalignant disease (eg, hydronephrosis, liverfailure, or other conditions) that could compromise protocol objectivesin the opinion of the investigator and/or the sponsor

13. Are currently receiving any other investigational agent

14. Have exhibited allergic reactions to a similar structural compound,biological agent, or formulation

15. Have undergone significant surgery to the gastrointestinal tractthat could impair absorption or that could result in short bowelsyndrome with diarrhea due to malabsorption

16. Have a history of severe adverse reaction (eg, hypersensitivityreaction, anaphylaxis) to sulfonamides

17. Patients scheduled to receive immunotherapy or TKI regimens plus thestudy drug must not be currently taking high-dose steroids (i.e.,physiologic dose approximately equivalent to 15 mg/day of prednisone)

Study Assessments (Predose)

Screening/Baseline Period (within 14 Days Prior to First Dose)

The following activities and evaluations are performed within 14 daysprior to administration of the first dose of study drug:

-   -   Signed Informed Consent    -   Collect and document a complete medical history including        histologically confirmed diagnosis of advanced metastatic or        progressive solid tumor and all other measures of disease and        disease symptoms including extent of tumor burden, radiographic        assessment, ie, computed tomography (CT) scan of the chest,        abdomen, pelvis; appropriate tumor markers (e.g., PSA, CA19-9).    -   Perform a full physical examination including height (cm) and        weight (kg)    -   Record vital signs (temperature, heart rate, systolic and        diastolic blood pressures)    -   Assess Eastern Cooperative Oncology Group (ECOG) Performance        Status (PS)    -   Perform a 12-lead electrocardiogram (ECG) including assessment        of QTcF interval    -   Collect blood for evaluation of laboratory parameters:        -   Hematology: complete blood count (CBC) with differential and            platelet count        -   Serum chemistries        -   Coagulation status: prothrombin time (PT) and activated            partial thromboplastin time (aPTT)    -   Collect urine sample for full urinalysis    -   Collect urine or serum sample for beta-human chorionic        gonadotropin (0-hCG) pregnancy test for females of child-bearing        potential    -   Perform pre-dose tumor biopsies in those patients enrolled in        the Biopsy Cohorts        -   The “Biopsy Cohorts” include patients enrolled in the five            expansion cohorts at the MTD who consent to undergo required            pre- and post-dose tumor biopsies (3 to 5 core samples            requested at each biopsy timepoint).    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements within        the past 14 days        Within 72 Hours Prior to First Dose of Cycle 1

The following activities and evaluations are performed anytime within 72hours prior to administration of the first dose (on Day 1) of studydrug:

-   -   Perform a full physical examination including weight (kg) and        calculation of BSA    -   Collect blood for evaluation of laboratory parameters:        Hematology and Serum chemistries    -   Collect urine or serum sample for β-hCG pregnancy test for        females of child-bearing potential    -   Collect blood for PBMCs, plasma, and serum for biomarker        assessments    -   Obtain archived tumor tissue    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Review all Inclusion/Exclusion criteria and determine if patient has metall eligibility criteria for inclusion into the study. Obtain MedicalMonitor (or designee) approval to enroll patient.

Study Treatment

The dosage of study drug (tartrate salt) is recalculated at thebeginning of each new treatment cycle to reflect changes in the bodysurface area (BSA) that may have occurred but will remain the same forall treatments within a treatment cycle. The dose is rounded down in theevent the calculated number is <0.5 to the nearest achievable dose basedon the amount of study drug in the capsules. The dose is rounded up tothe next level if the calculated dose is ≥0.5 to the nearest achievabledose based on available capsule strength. Patient doses are onlyadjusted if there is a ≥10% increase or decrease in body weight comparedto baseline.

Once the MTD has been determined in the expansion safety cohort, studydrug (di tartrate salt) is administered as a flat dose instead ofaccording to BSA.

Study drug is administered orally once daily for 21 days followed by 7drug free days (each cycle=28 days). Dosing may be repeated every cyclein the absence of disease progression or unacceptable toxicity. Studydrug should be taken in the morning after an overnight fast with up to200 mL or 7 fluid ounces of water at least 1 hour before ingesting anyfood or other medications.

Since PK and PD sampling is to be conducted during the first two cyclesof treatment, every effort is made to ensure timely transition fromCycle 1 to Cycle 2 (i.e., no extended delays between the cycles).

Evaluation of the safety and efficacy of study drug in this study occursin two phases: Phase 1a: Dose Escalation and Phase 1b: Expansion at MTDand Biopsy Cohorts

Phase 1a (Dose Escalation): Three-Patient Cohorts

The starting dose is 1.5 mg/m² for 21 out of 28 days in 3-patientcohorts using a standard 3+3 design. Once the first patient in eachcohort has completed 14 days of dosing with no DLTs, the second andthird patients are be enrolled simultaneously at the same dose. Once thelast patient enrolled has completed Day 28 without observation of a DLTand the next higher the compound of structure (I) dose level has not yetbeen studied, the dose is increased following a modified Fibonacci doseescalation scheme in a new 3-patient cohort according to the dose levelsprovided in Table 20.

TABLE 20 Dose Escalation Dose Proposed Daily Increment from No. ofPatients Level Dose Previous Dose^(a) Per Cohort −1^(b) 1 mg/m² −33% 3-6 1 1.5 mg/m² Starting Dose 3-6 2 3 mg/m² 100%  3-6 3 6 mg/m² 100% 3-6 4 9 mg/m² 50% 3-6 5 12 mg/m² 33% 3-6 6 16 mg/m² 33% 3-6 7 21 mg/m²33% 3-6 8 28 mg/m² 33% 3-6 9 37 mg/m² 33% 3-6 10  49 mg/m² 33% 3-611^(c ) 65 mg/m² 33% 3-6 ^(a)It is possible for additional and/orintermediate dose levels to be added during the course of the study.^(b)Dose level −1 represents treatment doses for patients requiring adose reduction from the starting dose level. It will also serve as alower dose level if the Starting Dose level is initially associated withunexpected or unacceptable toxicity. ^(c)If clinically indicated, doselevels higher than 65 mg/m² may be investigated.

If a DLT is observed in 1 of 3 patients at a given dose level, up to 3additional patients are enrolled and treated at that dose level. When upto 3 additional patients are added to a given dose level, if only 1 outof those 6 patients experiences a DLT, the dose is increased to the nextdose level. If ≥2 out of 3-6 patients at a dose level experience DLTs,the dose is decreased to the previous (lower) dose level and 3additional patients will be enrolled at that dose level.

If 0 or 1 patient in any of the 6 patients experience a DLT, but thenext higher dose level has already been studied, then the current doseis declared the MTD and the study advances to Phase 1b. The MTD isdefined as the dose at which ≤1 of 6 patients experience a DLT duringCycle 1 with the next higher dose having at least 2 of 3-6 patientsexperiencing a DLT during Cycle 1.

Phase 1 b: Expansion at MTD and Biopsy Cohorts

Once the MTD has been established, 5 additional cohorts of up to 20patients each (100 additional patients total) with the various diseasecharacteristics listed below are enrolled and treated in the Phase 1bstudy. Study drug is administered as a flat dose instead of according toBSA. Data collected from patients enrolled in each of these additionalcohorts is used to confirm safety, explore potential biomarkers, andevaluate potential signals of activity when study drug is administeredto specific groups of heavily pretreated patients or given incombination with immunotherapy or a TKI. Ten patients in each of these 5expansion cohorts are required to consent to undergo pre- and post-dosetumor biopsies. All patients who undergo these biopsies comprise the‘Biopsy Cohorts’. The five separate expansion cohorts enrolled andtreated at the MTD are described in the Patient Population sectionabove.

Management of Toxicides and Dosage Modification

Management of Toxicities

Adverse events may be treated with concomitant medications, as deemedclinically indicated by the Principal Investigator. All concomitantmedications are recorded in the source and on the appropriate CRF.

Adverse events that are moderate to severe in intensity and consideredPossibly, Probably, or Definitely related to study drug treatments mayresult in the termination of study treatment in the affected studypatient. Such termination is reviewed with the Sponsor's Medical Monitorat the earliest possible time. Following review with the Sponsor'sMedical Monitor, the study patient may be permanently withdrawn from thestudy depending upon the nature and severity of the event.

Dose-Limiting Toxicides (DLL)

A DLT is defined as any one of the following events observed withinCycle 1 regardless of attribution unless clearly and incontrovertiblyrelated to the underlying disease or extraneous causes (such asprogressive disease):

-   -   Grade 3 or greater febrile neutropenia    -   Grade 4 absolute neutrophil count (ANC) for ≥7 consecutive days    -   Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with        clinically significant bleeding or that requires a platelet        transfusion    -   Grade 3 or 4 non-hematologic AEs including nausea, vomiting,        diarrhea, and electrolyte imbalances persisting for more than 48        hours despite optimal medical management    -   Dosing delays≥2 weeks due to treatment emergent adverse events        or related severe laboratory test values        Dose Modifications

The dose of study drug is not reduced during Cycle 1. Doses of studydrug may be adjusted for patients who receive multiple cycles of studydrug. Dose reductions by one dose level is permitted based on theobserved toxicity that occurred during the preceding cycle. No dosere-escalations are allowed for any patient who had a previous dosereduction due to toxicity or delayed recovery. All dose modificationsneed to be discussed and approved with the medical monitor.

If a patient experiences toxicity, the patient may continue to receivestudy drug as defined in Table 21.

TABLE 21 Guide to Dose Adjustments Based on Toxicities Drug-Related AEAction Grade 1 Current dose level Grade 2 Investigator's option toreduce dose by 1 dose level with agreement of the Medical Monitor Grade3* Withhold, then reduce dose by 1 dose level upon recovery to ≤ Grade 1with agreement of the Medical Monitor. Grade 4 Investigator and MedicalMonitor review to determine if patient may continue on study withappropriate dose reduction upon recovery to ≤ Grade 1. *Excluding brief(based on the investigator's judgment) Grade 3 vomiting or diarrhea withsuboptimal management. Dose reduction to the next lower dose leveltested is performed initially. If further toxicities occur during one ormore cycles at the new reduce dose level, no further reductions arepermitted, and the patient should be discontinued from the study.

Patients who experience a DLT are required to discontinue studyparticipation, unless the Investigators and Medical Monitor determinethat it is in the best interest of the patient to continue with the dosereduction and only upon recovery of the toxicity to Grade 2 or better.

Dose reduction is required for patients who have a delay in treatmentgreater than 2 weeks due to a lack of recovery of any hematologic ornonhematologic toxicity, even if DLT criteria are not met. Subsequentretreatment of patients who are not able to be treated after a 2-weekdelay and who eventually recover is to be determined taking into accountthe potential benefit/risk for the individual patient. In addition, dosereductions are permitted for patients who have toxicities that do notmeet the criteria of a DLT.

For patients enrolled in the Phase 1b study receiving combinationtherapy (i.e., previous immunotherapy or TKI regimens plus study drug),modification of their immunotherapy or TKI regimen follow each approveddrug's full prescribing information.

Concomitant Medications and Therapies

Previous Therapies

Patients subject to the instant treatment may have undergone one or moreprevious therapies.

Concomitant Therapies

Concomitant therapies are any new or existing medications or therapytaken by the patient including:

-   -   Drugs, including but not limited to, prescription,        over-the-counter, birth control pills/patches/hormonal devices,        and homeopathic preparations    -   Nondrug therapies, including but not limited to,        thermal/laser/radiation procedures, vitamins, herbal        medicines/supplements.

During the Screening process (up to 14 days prior to anticipated firstdose of study drug), information on all concomitant therapies,medications, and procedures are recorded in the source documents andappropriate CRF along with the diagnosis or reason for use.

Once the patient receives the first dose of study drug, recording ofconcomitant therapies are limited to any new medication or modificationof an existing medication taken for treatment of an adverse event (AE).These therapies are recorded in the source documents and appropriate CRFalong with the diagnosis or reason for use. Those therapies used for thetreatment of an adverse event are to be linked to an AE anddocumentation of the AE must also be completed.

Permitted Therapies

Concomitant medications necessary for the health and well-being of thepatient and that do not interfere with study assessments are permittedduring the study at the Investigator's discretion. This includes the useof appropriate medications for the treatment of AEs and/or concurrentillnesses under the direction of the Principal Investigator. All suchtherapies must be recorded in the source and on the appropriate CRF.

Treatment with hematopoietic colony stimulating growth factors such asgranulocyte colony stimulating factor or granulocyte-macrophage colonystimulating factor is not initiated during Cycle 1 unless the patienthas experienced a DLT. Initiation of treatment witherythroid-stimulating agents may not occur during the first cycle oftherapy. If a patient has been on a steady dose of anerythroid-stimulating agent, they may continue to use the agent at thesame dose during Cycle 1 and later cycles.

Patients receiving immunotherapy plus study drug may receive concomitantsteroids for the treatment of immune response AEs at the investigator'sdiscretion.

Prohibited Therapies

The following medications are excluded from concomitant use by allpatients:

-   -   Anticancer therapies (chemotherapy, radiation therapy,        immunotherapy) within the month prior to the first study drug        administration and during the cycle of study treatment. Patients        with EGFR+ NSCLC or immunotherapy-resistant tumors who are        enrolled in the Phase 1b study continue treatment with their        previous immunotherapy or TKI regimens, respectively, and add        study drug.    -   CYP2C19 Metabolizers: Patients receiving CYP2C19 metabolizers        prior to study treatment should be monitored closely. If        possible, the investigator should cease patient's treatment with        a CYP2C19 substrate prior to first dose, or at a minimum, switch        to an alternative, but equivalent treatment that is not a        CYP2C19 metabolizer. If a patient must remain on a CYP2C19        metabolizer, treatment with study drug should proceed cautiously        and the patient observed closely throughout the duration of the        study.    -   Patients must not be taking H2-receptor antagonists such as        cimetidine, ranitidine, and famotidine, or any proton pump        inhibitors such as omeprazole, lansoprazole, esomeprazole and        pantoprazole. Patients must stop these medications within 7 days        prior to starting treatment.        Birth Control Requirements for Fertile Patients

Sexually active patients and their partners must use an effective methodof contraception associated with a low failure rate prior to study entryand for the duration of study participation and for 30 days after thelast dose of study drug. The following are considered effectivecontraceptives: (1) oral contraceptive pill; (2) condom plus spermicide;(3) diaphragm plus spermicide; (4) abstinence; (5) patient or partnersurgically sterile; (6) patient or partner more than 2 yearspost-menopausal; or (7) injectable or implantable agent/device.

Treatment Assessments

(i) Cycle 1

Day 1 (and as Otherwise Indicated):

-   -   Record vital signs (temperature, heart rate, systolic and        diastolic blood pressures) prior to first dose    -   Obtain baseline signs and symptoms prior to first dose    -   Perform 12-lead ECG including assessment of QTcF interval:    -   For Phase 1a patients: ECG time points to include just prior to        first dose, at 0.5, 1, 2 hours (±10-minute window for each time        point), and at 4 hours (±20-minute window) post dosing    -   For Phase 1b patients: ECG time points for the first 3 patients        enrolled in the immunotherapy-resistant and EGFR+ NSCFC cohorts        to include just prior to first dose; at 0.5, 1, and 2 hours        (±10-minute window for each time point); and 4 hours (±20-minute        window) post dosing.    -   All other Phase 1b patients: Perform 12-lead ECG including        assessment of QTcF interval pre-dose only    -   Collect blood for PBMCs, plasma, and serum for assessments        according to the schedule in Section 7.4    -   Collect blood for analysis of PK parameters

Daily on Days 1-21

-   -   Instruct patients to take study drug orally every day on Days        1-21    -   Instruct patients to record the date and time they took their        dose in their dosing diary

Weekly (Days 8, 15, 22 [±3 Days])

The following activities and evaluations are performed weekly (or asotherwise indicated) during Cycle 1:

-   -   On Day 8 and 22, collect blood for PBMCs, plasma, and serum for        biomarker assessments according to the schedule in Section 7.4    -   Perform an abbreviated physical examination    -   Record vital signs (temperature, heart rate, systolic and        diastolic blood pressures)    -   Collect blood for evaluation of laboratory parameters:        Hematology; Serum chemistries    -   On Days 8, 15, and 21, collect blood for analysis of PK        parameters    -   Assess for adverse events (AEs)    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

(ii) Cycle 2

Daily on Days 1-21

-   -   Instruct patients to take study drug orally every day on Days        1-21    -   Instruct patients to record the date and time they took their        dose in their dosing diary

Day 1

The following activities and evaluations are performed on Day 1 of Cycle2:

-   -   Perform a full physical examination including weight (kg) and        calculation of BSA    -   Assess ECOG PS    -   Collect urine or serum sample for β-hCG pregnancy test for        females of child-bearing potential    -   Perform 12-lead ECG just prior to dosing including assessment of        QTcF interval    -   Collect blood for PBMCs, plasma, and serum for biomarker        assessments    -   Collect blood for analysis of PK parameters

Weekly (Days 1, 8, 15, 22 [±3 Days])

The following activities and evaluations are performed weekly duringCycle 2:

-   -   Abbreviated physical examination (Days 8, 15, 22)    -   Record vital signs (temperature, heart rate, systolic and        diastolic blood pressures)    -   Collect blood for evaluation of laboratory parameters:        Hematology; Serum chemistries    -   On Days 8, 15, and 21, collect blood for analysis of PK        parameters according to the schedule in Section 7.3    -   Assess for adverse events (AEs)    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Day 28 (−4 Days):

-   -   Assess for response and tumor burden using RECIST v1.1 and        iRECIST including appropriate tumor markers using same methods        used at baseline    -   Perform post-dose tumor biopsies in those patients enrolled in        the Biopsy Cohorts. Ideally, the biopsy should be obtained from        the same general location as the pre dose biopsy, if feasible        and safe, and from a metastatic lesion and not the primary        tumor. Three to five (3 to 5) core samples are requested at each        biopsy timepoint.

(iii) Cycles≥3

Patients may continue to receive study drug in 28-day cycles at the samedose given during Cycle 1 until they experience unacceptable toxicity orunequivocal disease progression. Before proceeding with the next oddnumbered cycle, response and tumor burden must be assessed and confirmedfor continued response/clinical benefit.

Daily on Days 1-21

-   -   Instruct patients to take study drug orally every day on Days        1-21    -   Instruct patients to record the date and time they took their        dose in their dosing diary

Day 1

The following activities and evaluations are performed on Day 1 of Cycle3 and all subsequent cycles of treatment:

-   -   Perform a full physical examination including weight (kg) and        calculation of BSA    -   Assess ECOG PS    -   Collect urine or serum sample for β-hCG pregnancy test for        females of child-bearing potential    -   Perform 12-lead ECG just prior to dosing including assessment of        QTcF interval (Cycle 3 only)    -   Collect blood for PBMCs, plasma, and serum for biomarker        assessments    -   Collect blood for analysis of PK parameters (Cycle 3 only)

Days 1 and 15 (±3 Days)

The following activities and evaluations are performed every two weeksduring Cycle 3 and all subsequent cycles of treatment:

-   -   Abbreviated physical examination (Day 15)    -   Record vital signs (temperature, heart rate, systolic and        diastolic blood pressures)    -   Collect blood for evaluation of laboratory parameters:        Hematology; Serum chemistries    -   Assess for adverse events (AEs)    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Day 28 (−4 Days)

The he following activities and evaluations are performed on Day 28 (−4days) of every EVEN cycle (ie, Cycle 4, Cycle 6, etc): Assess forresponse and tumor burden using RECIST v1.1 and iRECIST includingappropriate tumor markers using same methods used at baseline.

Criteria for Evaluation

Safety Endpoints

Tolerance and toxicity of oral study drug are assessed throughevaluation of physical examinations, vital signs, laboratory studies,solicited and unsolicited adverse events including DLTs, and all causesof mortality.

Incidence rates of treatment-emergent adverse events are summarizedwithin study drug dose level at the MedDRA preferred term and primarysystem organ class levels. Similar summaries are made for subsets of AEssuch as (1) those judged by a physician to be related to studytreatment, and (2) serious adverse events (SAEs).

Other routine safety assessments (e.g., clinical laboratory parametersand vital signs) are summarized by study drug dose level using mean,standard deviation, median, minimum and maximum changes from baselinevalues.

Efficacy Endpoints

Any objective response to treatment with study drug is noted using theRECIST v1.1 (see, e.g., Eisenhauer et al. “New response evaluationcriteria in solid tumours: revised RECIST guideline (version 1.1)” Eur JCancer 2009, 45, 228-247) definitions of response or the iRECISTguidelines (see, e.g., Seymour et al. “RECIST working group. iRECIST:guidelines for response criteria for use in trials testingimmunotherapeutics.” Lancet Oncol 2017, 18, e143-e152) for patientsreceiving concomitant immunotherapy during the Phase 1b study. A summaryis provided below.

(i) Response Evaluation Criteria in Solid Tumors (RECIST), v1.1

Response criteria—evaluation of target lesions include:

-   -   Complete Response (CR): Disappearance of all target lesions. Any        pathological lymph nodes (whether target or nontarget) must have        reduction in short axis to <10 mm.    -   Partial Response (PR): At least a 30% decrease in the sum of        diameters of target lesions, taking as reference the baseline        sum diameters.    -   Progressive Disease (PD): At least a 20% increase in the sum of        diameters of target lesions, taking as reference the smallest        sum on study (this includes the baseline sum if that is the        smallest on study). In addition to the relative increase of 20%,        the sum must also demonstrate an absolute increase of at least        5 mm. (Note: the appearance of one or more new lesions in also        considered progression).    -   Stable Disease (SD): Neither sufficient shrinkage to qualify for        PR nor sufficient increase to qualify for PD taking as        references the smallest sum diameters while on study.

Response criteria—Evaluation of nontarget Lesions include:

-   -   Complete Response (CR): Disappearance of all non-target lesions        and normalization of tumor marker level. All lymph nodes must be        nonpathological in size (<10 mm short axis).    -   Non-CR/Non-PD: Persistence of one or more nontarget lesion(s)        and/or maintenance of tumor marker level above the normal        limits.    -   Progressive Disease (PD): Unequivocal progression of existing        nontarget lesions* (Note: the appearance of one or more new        lesions is also considered progression)

Although a clear progression of “nontarget” lesions only is exceptional,in such circumstances, the opinion of the treating physician shouldprevail and the progression status should be confirmed later on by thereview panel (or study chair).

(ii) Response Evaluation Criteria in Solid Tumors for Trials UsingImmunotherapy (iRECIST)

A phenomenon known as ‘pseudoprogression’ can occur in patients withsolid tumors receiving immunotherapy (i.e., checkpoint inhibitors andother immune-modulating agents). With pseudoprogression, tumor volumecan temporarily increase as a result of immune-cell infiltration ratherthan true disease progression. Members of the RECIST Working Group,along with input from several pharmaceutical companies and regulatoryagencies in the US and Europe, drafted the iRECIST guidelines forassessing changes in tumor burden in these patients. While similar totraditional RECIST guidelines, iRECIST requires additional imagingshowing continued tumor growth 4-to-8 weeks after initial evidence ofmounting tumor burden for the disease to be considered progressing(stable disease does not count as disease progression).

Pharmacokinetic Endpoints

Plasma concentrations of oral study drug are summarized by descriptivestatistics, including mean, n, standard deviation, coefficient ofvariation, minimum, maximum, and median. Prior to analysis of studysamples, the assay sensitivity, specificity, linearity, andreproducibility is documented.

Plasma PK analyses for oral study drug and known metabolites, if any,and dose proportionality will be determined on Days 1 and 21 of Cycle 1in all patients enrolled into the Phase 1a study as well as thoseenrolled into selected cohorts in the Phase 1b study.

The PK schedule is followed for all patients enrolled in the Phase 1astudy. In the Phase 1b study, this PK sampling schedule only applies topatients with EGFR+ NSCLC or immunotherapy-resistant tumors who areenrolled in expansion cohorts at the MTD and who receive study drug incombination with their previous anticancer therapies.

Pharmacodynamic Endpoints

PBMCs, plasma, and serum is collected to assess activity of study drugon predictive biomarkers including, but not limited to, GAS6 and AXL.

A predose tumor biopsy (within 14 days prior to first dose) and apost-dose tumor biopsy (Cycle 2, Day 28 [−4] days) is performed in 10patients enrolled in each of the 5 Phase 1b expansion cohorts (BiopsyCohorts) to assess activity of study drug on predictive biomarkers. If apredose biopsy is not possible, adequate archival tissue (3 to 5individual core samples collected within 4 weeks of screening) may besubmitted instead with sponsor approval. Ideally, the post-dose biopsyshould be obtained from the same general location as the predose biopsyif feasible and safe, and from a metastatic lesion and not the primarytumor. Three to five (3 to 5) individual core samples are requested ateach biopsy timepoint. Tumor tissue is examined using a variety oftechniques to assess changes in potential biomarkers after exposure tostudy drug. While these PD analyses are considered exploratory innature, they are evaluated in conjunction with pharmacokineticparameters, as well as any signals of efficacy and safety, to see ifthere is any correlation with changes in potential biomarkers.

Archived tumor tissue (primary and metastatic site[s], if available) isrequested from all patients to assess potential predictive biomarkers.

Adverse Events

An adverse event (AE) is defined as any untoward medical occurrenceassociated with the use of a drug in humans, whether or not considereddrug related. An AE can therefore be any unfavorable and unintended sign(including an abnormal laboratory finding), symptom, or diseasetemporally associated with the use of a drug, whether or not related tothe drug product.)

Suspected adverse reaction means any adverse event for which there is areasonable possibility that the drug caused the adverse event. For thepurposes of Investigational New Drug (IND) safety reporting, ‘reasonablepossibility’ means there is evidence to suggest a causal relationshipbetween the drug and the adverse event.

Unexpected adverse event or unexpected suspected adverse reaction: Anadverse event or suspected adverse reaction is considered unexpected ifit is not listed in the current Investigator's Brochure or is not listedat the specificity or severity that has been observed; or, is notconsistent with the risk information described in the generalinvestigational plan (clinical study protocol).

Toxicities are assessed according to the NCI CTCAE, version 4.03. Whenthe NCI CTCAE grade is not available, the investigator uses thefollowing toxicity grading: mild, moderate, severe, life threatening orfatal.

TABLE 21A Grades of Toxicities GRADE 1 - Mild: Transient or milddiscomfort; no limitation in activity; no medical intervention/ therapyrequired. GRADE 2 - Moderate: Mild to moderate limitation in activity -some assistance may be needed; no or minimal medicalintervention/therapy required. GRADE 3 - Severe: Marked limitation inactivity, some assistance usually required; medical intervention/therapyrequired, hospitalizations possible. Grade 4 - Life Extreme limitationin activity, significant Threatening assistance required; lifethreatening (immediate risk of death); significant medicalintervention/therapy required hospitalization or hospice case probable.Grade 5 - Fatal Results in death.

-   -   Relationship of the adverse event (AE) to the study drug should        be defined as follows:

Unrelated: AE is clearly not related to the investigational agent(s)Unlikely: AE is doubtfully related to the investigational agent(s)Possibly: AE may be related to the investigational agent(s) Probably: AEis likely related to the investigational agent(s) Definitely: AE isclearly related to the investigational agent(s)

A serious adverse event (SAE) is defined as any experience that suggestsa significant hazard, contraindication, side effect, or precaution. AnSAE includes:

-   -   Any death, or    -   Any life-threatening event (ie, the patient is at immediate risk        of death from the event as it occurred), or    -   Any event that is persistently, significantly, severely or        permanently disabling, or requires intervention to prevent such        disability, or    -   Any event which requires inpatient hospitalization or prolongs        hospitalization, or    -   Any congenital abnormality/birth defect, or    -   Any medically significant event that may jeopardize the patient        or may require intervention to prevent one of the other outcomes        listed above.

Example 49: Combined Phase 1/2 Study to Investigate the Safety,Pharmacokinetics, Pharmacodynamics, and Clinical Activity in Patientswith Previously Treated Chronic Lymphocytic Leukemia (CLL)/SmallLymphocytic Lymphoma (SLL)

The compound of structure (I) (described in Example 48 above) is a noveloral inhibitor that targets AXL kinase and reverses the mesenchymalphenotype associated with advanced cancers. The compound hasdemonstrated profound single agent activity in CLL B cells takendirectly from patients even if the patient has high risk factors (i.e.,17p/P53 deletions) or progressed on other agents (i.e., ibrutinib). Thisstudy is designed to identify the maximum tolerated dose (MTD), safetyprofile and recommended Phase 2 dose (RP2D) of study drug (described inExample 48 above) when administered orally once daily for 28 days on a28 day cycle to patients with previously treated CLL. In some instances,treatment cycles are repeated if the patient continues to show benefitand if study drug is reasonably well tolerated.

The study drug, in oral form, is supplied as a powder in hard gelatincapsules (size #3 for the 1-, 4-, 16-, and 25-mg doses; size #0 for the100-mg dose) and is manufactured under current Good ManufacturingPractices (cGMP) for investigational use.

Study drug capsules are formulated in 1-mg, 4-mg, 16-mg, 25-mg, and 100mg strengths and are packaged into round high-density polyethylenebottles with polyester coils as headspace fillers. Bottles are thenheat-sealed, fitted with child-resistant caps, and placed in low-densitypolyethylene bags as secondary packaging.

Ibrutinib, an approved pharmaceutical product, is provided bycommercially available sources. Ibrutinib capsules for oraladministration are supplied as white opaque capsules that contain 140 mgibrutinib as the active ingredient. Each white opaque capsule is markedwith “ibr 140 mg” in black ink.

The objectives for the Phase I trial include:

-   -   To characterize the safety and toxicity profile of study drug        when administered orally once daily for 28 days (each cycle is        28 days; no drug-free period) in the following patient groups:        -   Group 1 (study drug monotherapy): those with CLL/SLL who are            intolerant to, or have had progressive disease on B-cell            receptor antagonists, BCL-2 antagonists or other            investigational treatments for CLL/SLL        -   Group 2 (study drug and ibrutinib combination therapy):            those with CLL/SLL who have progressed on ibrutinib, yet the            treating provider considers continuation of ibrutinib            therapy to be in the best interest of the patient    -   To determine the RP2D of study drug when administered orally on        this schedule to the defined patient groups    -   To observe patients for any evidence of antileukemic activity of        oral study drug by determining the Objective Response Rate        ([ORR], ie, rate of complete response [CR] plus rate of partial        response [PR] in the defined patient groups according to        guidelines set forth by the 2018 International Workshop on CLL        (IWCLL)    -   To evaluate the pharmacokinetics (PK) of oral study drug in the        defined patient groups    -   To study potential biomarkers relevant to disease and        pharmacodynamics (PD) of oral study drug in the defined patient        groups through assessment of analytes including, but not limited        to, soluble AXL, AXL expression and phosphorylation, growth        arrest specific 6 (GAS6), and mesenchymal transcription factors        in peripheral blood samples and bone marrow

The Objectives for the Phase II trial include:

-   -   To determine the ORR in the two defined patient groups according        to guidelines set forth by the 2018 IWCLL    -   To determine the Duration of Response (DoR, ie, the time from        tumor response to disease progression)    -   To determine the Progression-free Survival (PFS, ie, the time        from first dose to objective tumor progression or death)    -   To determine the rate of Overall Survival (OS, ie, the time from        first dose to death from any cause)    -   To study potential biomarkers relevant to disease and        pharmacodynamics (PD) of oral study drug in the defined patient        groups through assessment of analytes including, but not limited        to, soluble AXL, AXL expression and phosphorylation, growth        arrest specific 6 (GAS6), and mesenchymal transcription factors        in peripheral blood samples and bone marrow        Justification for Treatment Plan

The starting dose is based on a thorough review of both the rat and dogGLP toxicology studies, as well as preliminary results from the Phase 1study in patients with advanced metastatic or progressive solid tumors(see Example 48). The compound of structure (I) was administered orallyonce each day for 28 days in the GLP toxicology studies. In the rat, allfindings were reversible or felt not to be clinically significant andthe NOAEL was determined as 2 mg/kg or 12 mg/m². Based on the animaltoxicology and safety studies, the starting dose for the compound ofstructure (I) was 1.5 mg/m²/day.

Given the clinical experience acquired in this solid tumor study and thelack of dose-limiting toxicities (DLTs) observed in any of the first 6cohorts, the starting dose for the tartrate salt of compound ofstructure (I) in Group 1 (monotherapy) is a 25-mg flat dose. Thestarting dose is approximately one dose level below the current Phase 1asolid tumor study (Example 48 above). Group 2 patients (combinationtherapy) start at one dose level below Group 1, or a 20-mg flat dose, toensure safety particularly with the combination of ibrutinib and thetartrate salt of compound of structure (I). The use of a flat, ratherthan body size-based dose, particularly for orally administered drugs,is preferable as it facilitates the use of the drug by patients andphysicians and reduces the number of dosage strengths needed, improvingcompliance.

Because the tartrate salt of compound of structure (I) has been welltolerated to date in the solid tumor study, the starting dose of 25 mgin the monotherapy arm is equivalent to 700 mg (25 mg×28 days, or 80% ofthe current dose in the solid tumor study). The starting dose in thecombination arm (i.e., the tartrate salt of compound of structure (I)plus ibrutinib) is 20 mg, which is a further 20% reduction from the 25mg starting dose in the monotherapy arm. The safety information from thesolid tumor study, together with the lower starting doses suggested inthis study, support the proposed continuous, 28-day dosing schedule.

Patent Population

Adult patients with chronic lymphocytic leukemia (CLL) and/or smalllymphocytic lymphoma (SLL) who:

-   -   1. are intolerant to, or have had progressive disease on B-cell        receptor antagonists, BCL-2 antagonists or other investigational        treatments for CLL/SLL (Group 1; monotherapy); or    -   2. have progression of disease on ibrutinib, yet the treating        provider considers continuation of ibrutinib therapy to be in        the best interest of the patient (Group 2; combination therapy        with ibrutinib).        Additional Inclusion Criteria

To be eligible for participation, patients must meet all of thefollowing inclusion criteria:

-   -   1. Be ≥18 years old    -   2. Have an established, pathologically confirmed diagnoses of        CLL/SLL requiring therapy according to the 2018 IWCLL guidelines    -   3. Have received at least one prior therapy for CLL/SLL and can        be classified in one of two patient groups:        -   Group 1 (monotherapy): Patients with CLL/SLL who are            intolerant to, or have progressed on B-cell receptor            antagonists and/or BCL-2 antagonists or        -   Group 2 (ibrutinib combination therapy): Patients with            CLL/SLL who have progression of disease on ibrutinib and the            treating provider considers continuation of ibrutinib            therapy to be in the best interest of the patient    -   4. Have an Eastern Cooperative Oncology Group (ECOG) performance        status≤2    -   5. Have adequate hematologic function: (a) Absolute neutrophil        count (ANC)≥500/μL; (b) Platelet count≥30,000/μL; (c)        Hemoglobin≥8 g/dL in the absence of transfusions within the        previous 2 weeks    -   6. Have adequate organ function: (a) Creatinine clearance≥30        mL/min; (b) Alanine aminotransferase (ALT)/aspartate        aminotransferase (AST) level≤2.5× upper limit of normal        (ULN); (c) Have a total bilirubin level≤1.5×ULN (unless        secondary to Gilbert syndrome, hemolysis, or leukemia)    -   7. Have acceptable coagulation status: Activated partial        thromboplastin (aPTT) and prothrombin time (PT)≤1.5×ULN    -   8. Have a negative pregnancy test (if female of childbearing        potential)    -   9. Be nonfertile or agree to use an adequate method of        contraception. Sexually active patients and their partners must        use an effective method of contraception (hormonal or barrier        method of birth control, or abstinence) prior to study entry and        for the duration of study participation and for at least 30 days        after the last study drug dose. Should a woman become pregnant        or suspect that she is pregnant while participating in this        study, she should inform her treating physician immediately.    -   10. Have read and signed the Institutional Review Board        (IRB)-approved informed consent form (ICF) prior to any study        related procedure. (In the event that the patient is rescreened        for study participation or a protocol amendment alters the care        of an ongoing patient, a new ICF must be signed.)    -   11. Be able to comply with the requirements of the entire study        Exclusion Criteria

Patients meeting any 1 of these exclusion criteria will be prohibitedfrom participating in the study.

-   -   1. Have undergone prior autologous or allogeneic stem cell        transplant within ≤3 months, have not recovered from transplant        associated toxicities, or requires graft versus host        immunosuppressive therapy    -   2. Have known central nervous system (CNS) involvement    -   3. Have Richter's transformation of CLL    -   4. Have received any monoclonal antibody therapy directed at        treatment of the patient's malignancy within 2 weeks prior to        anticipated first dose    -   5. Have received any anticancer therapy including chemotherapy,        radiotherapy, or an investigational anticancer drug within less        than 5 half-lives of the last dose of that treatment. This        exclusion criterion is not applicable to patients requiring        continuation on ibrutinib. (Note: Certain patients with a        rapidly rising white blood cell count while on ibrutinib may        need to remain on this drug for medical reasons. These patients        will need to be approved by the Medical Monitor and treated in        accordance with the protocol.)    -   6. Have received>20 mg/day of prednisone and 0.1 mg/day of        mineralocorticoids within 7 days prior to anticipated first dose    -   7. Have a corrected QT interval of >450 msec (males) and >470        msec (females) using Fridericia's correction formula    -   8. Have a significant history of renal, neurologic, psychiatric,        endocrinologic, metabolic, immunologic, hepatic, or        cardiovascular disease or any other medical condition that, in        the opinion of the Investigator, would adversely affect his/her        participation in the study    -   9. Are pregnant and/or nursing, or refuse to use appropriate        contraceptives during the course of the study and for at least        30 days after the last dose of study drug    -   10. History of another malignancy in the last 5 years except for        the following adequately treated: (a) Local basal cell or        squamous cell carcinoma of the skin; (b) Carcinoma in situ of        the cervix or breast; (c) Papillary, noninvasive bladder        cancer; (d) Early stage prostate cancer for which observation is        clinically indicated; (e) Other Stage 1 or 2 cancers currently        in complete remission (f) Any other cancer that has been in        complete remission for 2 years or surgically cured. Medical        Monitor may be contacted for additional determination of        acceptable prior cancer history    -   11. Have known gastrointestinal disorders (eg, malabsorption        syndrome), complications (eg, dysphagia), or surgery that could        make consumption or absorption of oral medications problematic    -   12. Have an uncontrolled systemic infection (viral, bacterial,        or fungal) or fever and neutropenia within 7 days prior to        anticipated first dose    -   13. Have active and uncontrolled autoimmune cytopenias for 2 or        more weeks including autoimmune hemolytic anemia or idiopathic        thrombocytopenic purpura (ITP)    -   14. Have received prior therapy with an AXL inhibitor    -   15. Have exhibited allergic reactions to a similar structural        compound, biological agent, or formulation    -   16. Are unwilling or unable to comply with procedures required        in this protocol    -   17. Have a history of severe adverse reaction (eg,        hypersensitivity reaction, anaphylaxis) to sulfonamides        Treatment Schedule and Dosing

This is a combined Phase 1/2 study of oral study drug in patients withpreviously treated CLL/SLL. In both Phase 1 and Phase 2, studyparticipants are assigned to 1 of 2 groups:

-   -   Group 1 (study drug monotherapy): Patients who are intolerant        to, or have had progressive disease on B-cell receptor        antagonists and/or BCL-2 antagonists or other investigational        treatments.    -   Group 2 (study drug and ibrutinib combination therapy): Patients        who have progression of disease on ibrutinib and the treating        provider considers continuation of ibrutinib therapy to be in        the best interest of the patient.

Both groups of patients are treated identically with study drug andundergo the same study assessments. The study may take up to 36 monthsto enroll up to 108 patients (up to 27 patients in each group (Group 1and Group 2) in both Phase 1 (n=54) and Phase 2 (n=54).

Study drug is administered orally once daily for 28 days (each cycle is28 days; no drug-free period). Dosing may be repeated every cycle in theabsence of disease progression or unacceptable toxicity. Study drugshould be taken in the morning after an overnight fast with up to 200 mLor 7 fluid ounces of water at least 1 hour before ingesting any food orother medications. Administer ibrutinib orally once daily atapproximately the same time each day. Swallow the capsules whole withwater. Do not open, break, or chew the capsules.

Phase 1

Patients are enrolled in Group 1 and Group 2 in cohorts of 3 to 6patients simultaneously. Group 2 start at 1 dose level below the group 1starting dose. In each group, escalation of the study drug dose followsa standard 3+3 design with sequential cohorts of 3 patients treated withincrementally higher doses of study drug until a DLT is observed and theMTD is established. Once the first patient at a dose level is enrolled,the second and third patients are enrolled after 3 weeks if the initialpatient has not experienced a DLT or any unacceptable toxicity.

If 1 of 3 patients in a cohort experiences a DLT, up to 3 additionalpatients are treated at that dose level. If no additional DLTs areobserved in the expanded 3- to 6 patient cohort within 28 days after thelast patient was first dosed, the dose is escalated in a new cohort of 3patients. If 2 or more of 3 to 6 patients at a given dose levelexperience a DLT during the first cycle, then the MTD is exceeded and upto a total of 6 patients will be treated at the previous lower doselevel. If 0 or 1 of 6 patients experiences a DLT at this previous lowerdose level, this dose is declared the MTD.

The MTD is defined as the dose at which ≤1 of 6 patients experience aDLT during Cycle 1 with the next higher dose having at least 2 of 3 to 6patients experiencing a DLT during Cycle 1. Once the MTD or preliminaryRP2D is identified, an expansion cohort of up to six patients isenrolled in each patient group to confirm safety/confirm the suitabilityof the preliminary RP2D, to collect additional biomarker data, and tofurther explore efficacy. Up to 27 patients may be enrolled in eachpatient group for a total of up to 54 patients in Phase 1.

Additional dose levels, schedules or disease indications of study drugmay be explored, as appropriate, based on the modulation of keybiomarkers, the safety profile and clinical signals of activity.

Group 1 (study drug monotherapy): The starting dose for study drug inGroup 1 (monotherapy) is a 25-mg flat dose. The study drug isadministered orally once daily for 28 days (each cycle is 28 days; nodrug-free period). Patients may continue to receive study drug in 28-daycycles at the same dose given during Cycle 1 until they experienceunacceptable toxicity or unequivocal disease progression. Nointrapatient escalation of the study drug dose is permitted.

Group 2 (study drug in combination with ibrutinib): The starting dose ofstudy drug is a 20-mg flat dose. Study drug is administered orally oncedaily for 28 days (each cycle is 28 days; no drug-free period). Patientsalso receive ibrutinib at the same dose that they were receivingimmediately prior to study enrollment. Patients continue with thecombination of ibrutinib and study drug for at least 3 months afterstudy start. After that time, patients either continue with combinationtherapy or discontinue ibrutinib and continue with study drugmonotherapy at the discretion of the Investigator and in consultationwith the Medical Monitor. Patients may continue to receive study drug in28-day cycles at the same dose given during Cycle 1 until theyexperience unacceptable toxicity or unequivocal disease progression.

Ibrutinib may be stopped and reinitiated at the discretion of theInvestigator and in consultation with the Medical Monitor; however, thetotal time patients may receive treatment with ibrutinib is 2 years.

The escalated doses of study drug in the Phase I trial are provided inTable 22 below:

TABLE 22 Dose Escalation Dose Proposed Daily Increment from No. ofPatients Level Dose Previous Dose^(a) Per Cohort Group 1 (Monotherapy) 125 mg Starting Dose 3-6 2 33 mg 33% 3-6 3 45 mg 36% 3-6 4 58 mg 29% 3-65 75 mg 29% 3-6  6^(b) 100 mg 33% 3-6 Group 2 (Combination) 1 20 mgStarting Dose 3-6 2 25 mg 25% 3-6 3 33 mg 33% 3-6 4 45 mg 36% 3-6 5 58mg 29% 3-6  6^(b) 75 mg 29% 3-6  7^(b) 100 mg 33% 3-6 ^(a)It is possiblefor additional and/or intermediate dose levels to be added during thecourse of the study. ^(b)If clinically indicated, dose levels higherthan 100 mg may be investigated.Phase 2

In Phase 2, patients are enrolled in Group 1 and Group 2 based on theSimon 2 stage design. In Stage 1, up to 13 patients are enrolled intoeach patient group (total of 26 patients). If there are zero responsesamong these 13 patients in each group, the study is stopped. Otherwise,Stage 2 enrolls 14 additional patients in each group for a total of 27patients per group. If 4 or more responses are observed among 27patients, the conclusion is that the study treatment is worthy offurther investigation. When the true response rate of 20% (alternativehypothesis) is tested against the null hypothesis response rate of 5%,this design yields a Type I error rate of 0.05 and power of 80%.

Group 1 (study drug monotherapy): The starting dose is the RP2Ddetermined during Phase 1. Study drug is administered orally once dailyfor 28 days (each cycle is 28 days; no drug-free period). Dosing withstudy drug may continue until a patient experiences unacceptabletoxicity or unequivocal disease progression

Group 2 (study drug in combination with ibrutinib): The starting dose isthe RP2D determined during Phase 1. Study drug is administered orallyonce daily for 28 days (each cycle is 28 days; no drug-free period).Patients will also receive ibrutinib at the same dose that they werereceiving immediately prior to study enrollment.

Patients should continue with the combination of ibrutinib and studydrug for at least 3 months after study start. After that time, patientseither continue with combination therapy or discontinue ibrutinib andcontinue with study drug monotherapy at the discretion of theInvestigator and in consultation with the Medical Monitor. Patients maycontinue to receive study drug in 28-day cycles at the same dose givenduring Cycle 1 until they experience unacceptable toxicity orunequivocal disease progression.

Ibrutinib may be stopped and reinitiated at the discretion of theInvestigator and in consultation with the Medical Monitor; however, thetotal time patients may receive treatment with ibrutinib is 2 years.

Management of Toxicities and Dosage Modification

Management of Toxicities

AEs may be treated with concomitant medications, as deemed clinicallyindicated by the Principal Investigator. All concomitant medicationsmust be recorded in the source and on the appropriate electronic casereport form (eCRF).

AEs that are moderate to severe in intensity for NCI CTCAE toxicitygrading) and considered Possibly, Probably, or Definitely related tostudy drug treatments may result in the termination of study treatmentin the affected study patient. A patient may be permanently withdrawnfrom the study depending upon the nature and severity of the event.

Dosing-Limiting Toxicides (DLLs)

A DLT is defined as any one of the following events observed withinCycle 1, regardless of attribution unless clearly and incontrovertiblyrelated to the underlying disease or extraneous causes (such asprogressive disease; other decreases in the white blood cell count, orin circulating granulocytes, are not to be considered, since a decreasein the white blood cell count is a desired therapeutic endpoint):

-   -   Any Grade≥3 nonhematologic toxicity    -   Any Grade 3 AE that does not resolve to ≤Grade 1 within 72 hours        with use of supportive care    -   Any AST and ALT elevation≥5×ULN accompanied by serum bilirubin        levels>2×ULN    -   Any Grade≥3 electrolyte disturbances (eg, hyperkalemia,        hypophosphatemia, hyperuricemia) that do not resolve within <72        hours    -   Any Grade≥3 elevations in creatinine    -   Any Grade 5 toxicity    -   Any instance of febrile neutropenia        Study Drug Dose Modification

The dose of study drug is not reduced during Cycle 1. Doses of studydrug may be adjusted for patients who receive multiple cycles of studydrug. Dose reductions by one dose level is permitted based on theobserved toxicity that occurred during the preceding cycle. No dosere-escalations are allowed for any patient who had a previous dosereduction due to toxicity or delayed recovery.

If a patient experiences toxicity, the patient may continue to receivestudy drug as defined in Table 23 in conjunction with the guidelines setforth by the 2018 IWCLL Grading Scale for Hematologic Toxicity.

TABLE 23 Guide to Dose Adjustments Based on Toxicities Drug-Related AEAction Grade 1 Current dose level Grade 2 Investigator's option toreduce dose by 1 dose level with agreement of the Medical Monitor Grade3^(a) Withhold, then reduce dose by 1 dose level upon recovery to ≤Grade 1 with agreement of the Medical Monitor. Grade 4 Investigator andMedical Monitor review to determine if patient may continue on studywith appropriate dose reduction upon recovery to ≤ Grade 1.^(a)Excluding brief (based on the Investigator's judgment) Grade 3vomiting or diarrhea with suboptimal management.

Dose reduction to the next lower dose level tested are performedinitially. If further toxicities occur during 1 or more cycles at thenew reduced dose level, no further reductions are permitted, and thepatient is discontinued from the study.

Patients who experience a DLT are required to discontinue studyparticipation, unless the Investigators and Medical Monitor determinethat it is in the best interest of the patient to continue with the dosereduction and only upon recovery of the toxicity to Grade 2 or better.

Dose reduction are required for patients who have a delay in treatmentgreater than 2 weeks due to a lack of recovery of any hematologic ornonhematologic toxicity, even if DLT criteria are not met. In addition,dose reductions are permitted for patients who have toxicities that donot meet the criteria of a DLT. These toxicities are discussed todetermine if it would be in the best interest of the patient to continueto receive the compound of structure (I) at the next previous doselevel.

Ibrutinib Dose Modifications

The package insert for ibrutinib therapy should be followed by thetreating physician. As per the ibrutinib label: Interrupt ibrutinibtherapy for any Grade 3 or greater non-hematological, Grade 3 or greaterneutropenia with infection or fever, or Grade 4 hematologicaltoxicities. Once the symptoms of the toxicity have resolved to Grade 1or baseline (recovery), ibrutinib therapy may be reinitiated at thestarting dose. If the toxicity reoccurs, reduce dose by one capsule (140mg per day). A second reduction of dose by 140 mg may be considered asneeded. If these toxicities persist or recur following two dosereductions, discontinue ibrutinib. Recommended dose modifications aredescribed below in Table 24.

TABLE 24 Recommended Dose Modifications for Ibrutinib ToxicityOccurrence CLL Dose Modification First Hold ibrutinib until recovery toGrade ≤1 or baseline; May restart at 420 mg daily Second Hold ibrutinibuntil recovery to Grade ≤1 or baseline; May restart at 280 mg dailyThird Hold ibrutinib until recovery to Grade ≤1 or baseline; May restartat 140 mg daily Fourth Discontinue ibrutinib

If ibrutinib is interrupted for a reason other than toxicity (eg,unrelated illness) the first instance of interruption must be restartedwithin 42 days. Subsequent study medication interruptions lasting morethan 42 days, ibrutinib should be discontinued permanently.

Concomitant Medications and Therapies

Patients subject to the treatment disclosed in this Example may haveprevious therapies of the corresponding cancer.

Patients subject to the instant treatment may have one or moreconcomitant therapies, which are any new or existing medications ortherapy taken by the patient. Examples include:

-   -   Drugs, including but not limited to, prescription,        over-the-counter, birth control pills/patches/hormonal devices,        and homeopathic preparations    -   Nondrug therapies, including but not limited to,        thermal/laser/radiation procedures, vitamins, herbal        medicines/supplements.

Once the patient receives the first dose of the Study Drug, recording ofconcomitant therapies is limited to any new medication or modificationof an existing medication taken for treatment of an AE. These therapiesare recorded in the source documents and appropriate eCRF along with thediagnosis or reason for use. Those therapies used for the treatment ofan AE are to be linked to an AE and documentation of the AE must also becompleted.

Concomitant medications necessary for the health and well-being of thepatient and that do not interfere with study assessments are permittedduring the study. This includes the use of appropriate medications forthe treatment of AEs and/or concurrent illnesses.

Treatment with hematopoietic colony stimulating growth factors such asgranulocyte colony stimulating factor or granulocyte-macrophage colonystimulating factor may not be initiated during Cycle 1 unless thepatient has experienced a DLT. Initiation of treatment with erythroidstimulating agents may not occur during the first cycle of therapy. If apatient has been on a steady dose of an erythroid stimulating agent,they may continue to use the agent at the same dose during Cycle 1 andlater cycles.

Supportive care includes:

-   -   Careful monitoring of patients at high risk for tumor lysis        syndrome (TLS) (i.e., patients with any lymph node [LN]≥10 cm,        or absolute lymphocyte count [ALC]≥25×109/L and any LN≥5 cm) by        collection of blood and real-time (STAT) review of TLS        laboratory parameters (i.e., uric acid, potassium, phosphate,        calcium, and creatinine) on Day 1 of Cycle 1 at baseline        (predose) and at 6 hours and 24 hours post dose.    -   Infection Prevention (ie, prophylactic antibiotic, antiviral,        and/or antifungal therapy) to be initiated according to each        institution's standardized protocols

The following medications are excluded from concomitant use:

-   -   Anticancer therapies (chemotherapy, radiation therapy,        immunotherapy) within less than 5 half-lives of the last dose of        that treatment. Patients enrolled in Group 2 (study drug in        combination with ibrutinib) continue treatment with the        combination for at least 3 months. Ibrutinib may be stopped and        reinitiated; however, the total time patients may receive        treatment with ibrutinib is 2 years.    -   CYP2C19 Metabolizers: Patients who are known abnormal        metabolizers of CYP2C19 (i.e., extensive or poor) prior to study        treatment should be monitored closely. If possible, patient's        treatment with a CYP2C19 substrate should be terminated prior to        first dose, or at a minimum, switch to an alternative, but        equivalent treatment that is not a CYP2C19 substrate (inhibitor        or inducer). If a patient must remain on a CYP2C19 substrate,        treatment with study drug should proceed cautiously and the        patient observed closely throughout the duration of the study.    -   Group 2 patients (i.e., study drug in combination with        ibrutinib) should avoid co-administration of strong or moderate        CYP3A inhibitors (e.g., carbamazepine, rifampin, phenytoin, and        St. John's Wort) as these substances may increase ibrutinib        plasma concentrations.    -   Group 2 patients should avoid co-administration of strong CYP3A        inducers as these substances may decrease ibrutinib        concentrations.    -   Patients must not be taking H2-receptor antagonists such as        cimetidine, ranitidine, and famotidine, or any proton pump        inhibitors such as omeprazole, lansoprazole, esomeprazole and        pantoprazole. Patients must stop these medications within 7 days        prior to starting treatment.

Sexually active patients and their partners must use an effective methodof contraception associated with a low failure rate prior to study entryand for the duration of study participation and for 30 days after thelast dose of study drug. The following are considered effectivecontraceptives: (1) oral contraceptive pill; (2) condom plus spermicide;(3) diaphragm plus spermicide; (4) abstinence; (5) patient or partnersurgically sterile; (6) patient or partner more than 2 yearspostmenopausal; or (7) injectable or implantable agent/device.

Safety and Efficacy Evaluations

Predose Assessments

The following procedures and evaluations are performed within 14 daysprior to administration of the first dose of the Study Drug, after theICF is signed unless otherwise noted:

-   -   Collect and document a complete medical history including        histologically confirmed diagnosis of CLL/SLL. Examples include:        -   Perform a full physical examination, including height (cm)            and weight (kg) and review the following constitutional            symptoms suggestive of active disease        -   Unintentional weight loss≥10% within previous 6 months        -   Marked fatigue        -   Fevers≥100.5° F. or (38.0° C.) for ≥2 weeks without evidence            of infection        -   Night sweats for ≥1 month without evidence of infection    -   Record vital signs (body temperature, respirations, heart rate,        blood pressure)    -   Assess ECOG Performance Status    -   Evaluate laboratory parameters, including full serum chemistry,        hematology (complete blood count [CBC] with differential and        platelet count), coagulation parameters (PT and aPTT),        urinalysis, serum immunoglobulins, direct antiglobulin, and        serum β2-microglobulin    -   Perform a 12-lead ECG including assessment of corrected QT        interval (using Fridericia's correction formula) (QTcF)    -   Pregnancy test (urine or serum beta-human chorionic gonadotropin        pregnancy test for females of childbearing potential)    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements within        the past 14 days    -   Assess baseline disease status per 2018 IWCLL guidelines (within        28 days of Cycle 1 Day1):        -   Perform a computed tomography (CT) scan of neck, chest,            abdomen, and pelvis for evaluation of lymphadenopathy,            hepatomegaly, and splenomegaly;        -   Bone marrow biopsy and aspirate with matched peripheral            blood sample        -   The following should be obtained (within 28 days of Cycle 1            Day 1): molecular cytogenetics (FISH) for del(13q),            del(11q), del(17p), add(12) [peripheral blood]; karyotyping            with CpG (or institutional standard) stimulation [bone            marrow]; TP53 mutation analysis [peripheral blood]; and/or            immunoglobulin heavy-chain variable (IGHV) mutational            analysis [peripheral blood]    -   Positron emission tomography (PET) scan to assess for possible        Richter's transformation (within 14 days of first dose)

The following baseline procedures and evaluations are performed any timewithin 3 days (72 hours) prior to administration of the first dose ofstudy drug (not required to be repeated at Cycle 1 Day 1 if screeningexams are within 3 days prior to first dose):

-   -   Full physical examination, including weight (kg)    -   Record vital signs (body temperature, respirations, heart rate,        blood pressure)    -   Evaluate laboratory parameters: full serum chemistry; hematology        (CBC with differential and platelet count); pregnancy test        (urine or serum beta-human chorionic gonadotropin pregnancy test        for females of childbearing potential)    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements    -   Review all inclusion/exclusion criteria and determine if patient        has met all eligibility criteria for inclusion in the study.        Obtain Medical Monitor (or designee) approval to enroll patient.        Treatment Assessments

(i) Cycle 1

Day 1

-   -   Full physical examination, including weight (kg)    -   Record vital signs (temperature, respirations, heart rate, blood        pressure) prior to first dose    -   Obtain baseline signs and symptoms prior to first dose    -   Assess ECOG Performance Status    -   Evaluate laboratory parameters: full serum chemistry; hematology        (CBC with differential and platelet count); TLS labs to be        assessed at baseline (predose) and at 6 hours and 24 hours post        dose (real-time [STAT] review) in patients at high risk for TLS        (ie, patients with any LN≥10 cm, or ALC≥25×109/L and any LN≥5        cm); and/or pregnancy test (urine or serum beta-human chorionic        gonadotropin pregnancy test for females of childbearing        potential)    -   Perform 12-lead ECG just prior to first dose, including        assessment of QTcF    -   Collect blood for analysis of PK parameters in Phase 1 only    -   Collect blood for exploratory biomarker assessments    -   Assess for AEs    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Daily on Days 1 Through 28

-   -   Instruct patients to take study drug and ibrutinib (for Group 2        patients) orally every day on Days 1 through 28    -   Instruct patients to record the date and time they took their        dose(s) in their dosing diary

Weekly (Days 8, 15, and 22 [±3 Days])

The following activities and evaluations are performed weekly (or asotherwise indicated) during Cycle 1:

-   -   Perform an abbreviated physical examination (AE- or symptom        directed)    -   Record vital signs (temperature, respirations, heart rate, blood        pressure)    -   Evaluate laboratory parameters: full serum chemistry; hematology        (CBC with differential and platelet count)    -   On Day 8, collect blood for exploratory biomarker    -   Assess for AEs    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Day 28

On Day 28, collect blood for analysis of PK parameters according to theschedule in Section 7.3 in Phase 1 only

(II) Cycle 2

Day 1

-   -   Full physical examination, including weight (kg)    -   Record vital signs (temperature, respirations, heart rate, blood        pressure)    -   Evaluate laboratory parameters: full serum chemistry; hematology        (CBC with differential and platelet count); pregnancy test        (urine or serum beta-human chorionic gonadotropin pregnancy test        for females of childbearing potential)    -   Assess ECOG Performance Status    -   Perform 12-lead ECG just prior to dosing including assessment of        QTcF    -   Collect blood for exploratory biomarker assessments    -   Assess for AEs    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Daily on Days 1 Through 28

-   -   Instruct patients to take study drug and ibrutinib (for Group 2        patients) orally every day on Days 1 through 28    -   Instruct patients to record the date and time they took their        dose(s) in their dosing diary

Day 15 [±3 Days]

-   -   Perform an abbreviated physical examination (AE- or symptom        directed)    -   Record vital signs (temperature, respirations, heart rate, blood        pressure)    -   Evaluate laboratory parameters: full serum chemistry; hematology        (CBC with differential and platelet count)    -   Assess for AEs    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Day 28 (−4 Days)

-   -   Disease assessment—Assess for response per 2018 IWCLL        guidelines, including review of the following constitutional        symptoms suggestive of active disease: unintentional weight        loss≥10% within previous 6 months, marked fatigue;        fevers≥100.5° F. or (38.0° C.) for ≥2 weeks without evidence of        infection, and/or night sweats for ≥1 month without evidence of        infection; and/or CT scan of neck, chest, abdomen, and pelvis        evaluation of lymphadenopathy, hepatomegaly, and splenomegaly    -   If clinical and laboratory results indicate possible CR: collect        bone marrow and aspirate with matched peripheral blood for CBC        and determination of MRD (central lab assessment)

(iii) Cycles≥3

Patients may continue to receive study drug in 28-day cycles at the samedose given during Cycle 1 until they experience unacceptable toxicity orunequivocal disease progression.

Day 1

-   -   Perform the following activities and evaluations on Day 1 of        Cycle 3 and all subsequent cycles of treatment:        -   Full physical examination, including weight (kg)        -   Record vital signs (temperature, respirations, heart rate,            blood pressure)        -   Evaluate laboratory parameters: full serum chemistry;            hematology (CBC with differential and platelet count);            and/or pregnancy test (urine or serum beta-human chorionic            gonadotropin pregnancy test for females of childbearing            potential)        -   Assess ECOG Performance Status        -   Perform 12-lead ECG just prior to dosing including            assessment of QTcF        -   Collect blood for analysis of PK parameters        -   Collect blood for exploratory biomarker        -   Assess for AEs        -   Record all concomitant medications including all            prescription drugs, nonprescription drugs, and nutritional            supplements

Daily on Days 1 Through 28

-   -   Instruct patients to take study drug and ibrutinib (for Group 3        patients) orally every day on Days 1 through 28    -   Instruct patients to record the date and time they took their        dose(s) in their dosing diary

Day 15 (±3 Days)

The following activities and evaluations are performed during Cycle 3and all subsequent cycles of treatment:

-   -   Perform an abbreviated physical examination (AE- or symptom        directed)    -   Record vital signs (temperature, respirations, heart rate, blood        pressure)    -   Evaluate laboratory parameters: full serum chemistry; and/or        hematology (CBC with differential and platelet count)    -   Assess for AEs    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements

Day 28 (−4 Days)

The following evaluations are performed on Day 28 of every EVEN cycle(i.e., Cycle 4, Cycle 6, etc):

-   -   Disease assessment—Assess for response per 2018 IWCLL        guidelines, including review of the following constitutional        symptoms suggestive of active disease: unintentional weight        loss≥10% within previous 6 months, marked fatigue;        fevers≥100.5° F. or (38.0° C.) for ≥2 weeks without evidence of        infection, and/or night sweats for ≥1 month without evidence of        infection; and/or CT scan of neck, chest, abdomen, and pelvis        evaluation of lymphadenopathy, hepatomegaly, and splenomegaly    -   If clinical and laboratory results indicate possible CR: collect        bone marrow and aspirate with matched peripheral blood for CBC        and determination of MRD (central lab assessment)        End-of-Treatment Assessments

If, at any time, a patient discontinues study treatment, a visit isscheduled as soon as possible and within 14 days of the last dose ofstudy drug or within 14 days of the decision to discontinue studytreatment. If the decision to withdraw the patient occurs at a regularlyscheduled visit, that visit may become the End-of-Study visit ratherthan having the patient return for an additional visit.

-   -   Perform a full physical examination including weight (kg)    -   Record vital signs (temperature, respirations, heart rate, blood        pressure)    -   Evaluate laboratory parameters: full serum chemistry; hematology        (CBC with differential and platelet count); and/or pregnancy        test (urine or serum beta-human chorionic gonadotropin pregnancy        test for females of childbearing potential)    -   Assess ECOG Performance Status    -   Perform a 12-lead ECG including assessment of QTcF    -   Disease assessment—Assess for response per 2018 IWCLL        guidelines, including review of the following constitutional        symptoms suggestive of active disease: unintentional weight        loss≥10% within previous 6 months, marked fatigue;        fevers≥100.5° F. or (38.0° C.) for ≥2 weeks without evidence of        infection, and/or night sweats for ≥1 month without evidence of        infection; and/or CT scan of neck, chest, abdomen, and pelvis        evaluation of lymphadenopathy, hepatomegaly, and splenomegaly    -   If clinical and laboratory results indicate possible CR: collect        bone marrow and aspirate with matched peripheral blood for CBC        and determination of MRD (central lab assessment)    -   Collect blood for exploratory biomarker assessments    -   Assess for AEs    -   Record all concomitant medications including all prescription        drugs, nonprescription drugs, and nutritional supplements        Criteria for Evaluation        Safety Endpoints

Phase 1

Safety is monitored from the time of the first dose until 30 days afterthe last dose of study drug. During Phase 1, the safety endpoints areevaluated after Cycle 1. The dose escalation committee, comprised ofInvestigators, sponsor and CRO representatives, has access to completesafety profiles of all patients receiving study drug to enable decisionmaking.

The primary safety endpoint is to assess the tolerance and toxicity ofcontinuous orally administered study drug through evaluation of physicalexaminations, vital signs, laboratory parameters, solicited andunsolicited AEs including DLTs, and all causes of mortality up to 30days from the last dose in both phases of the study. In Phase 2, allcauses of mortality are also evaluated at 60 days from the lastadministered dose.

Overall safety profile is characterized by type, frequency, severity,seriousness, timing, duration, and relationship of study drug to AEs andlaboratory abnormalities. Treatment-emergent AEs (TEAEs), namely, AEswith initial onset or that worsen in severity after the first dose ofstudy drug will be classified using the Medical Dictionary forRegulatory Activities (MedDRA) v20.0 or higher and graded according toNCI CTCAE v5.0. All DLTs will be reported and the MTD and RP2Didentified.

Efficacy Endpoints

Phase 2

The primary efficacy endpoint of the Phase 2 portion of the study is todetermine the ORR (rate of CR or PR) in patients with previously treatedCLL/SLL according to IWCLL guidelines 2018 (see, e.g., Hallek et al.“Guidelines for Diagnosis, Indications for Treatment, ResponseAssessment and Supportive Management of Chronic Lymphocytic Leukemia”(an ppdate of the NCI-sponsored guidelines from the InternationalWorkshop on Chronic Lymphocytic Leukemia)).

The secondary efficacy endpoints include:

-   -   DoR, defined as the time from documentation of tumor response to        disease progression.    -   PFS, defined as the time from study enrollment until objective        tumor progression or death.    -   OS, defined as the time from study enrollment to death from any        cause.        Efficacy assessments are performed at Cycle 2/Day 28 and then        every even cycle (Cycle 4/Day 28, Cycle 6/Day 28, etc). Response        rates are calculated in Stage 1 and Stage 2 as per the Simon 2        stage design.

A DSMB is monitor key outcomes from the study during the Phase 2 portionof the study.

Disease response is assessed at every 2 cycles per 2018 IWCLL guidelines

Pharmacokinetic Endpoints

Plasma PK analysis of oral study drug is performed in Cycle 1 on Days 1and 28 in all patients enrolled in the Phase 1 portion of this study.Known metabolites of study drug, if any, may also be evaluated. Standardplasma PK parameters are calculated, including: Cmax, Tmax, AUC fromtime 0 to 24 hours (AUC0-24), AUC0 inf, AUC from time 0 to time t(AUCt), half-life (t½), and clearance using noncompartmental methods(CL). If data permit, dose proportionality and accumulation ratio areestimated in Phase 1 Cycle 1. PK samples should be drawn on theprotocol-specified day.

Plasma concentrations of oral study drug are summarized by descriptivestatistics, including mean, n, standard deviation, coefficient ofvariation, minimum, maximum, and median. A validated bioanalyticalmethod for the detection of study drug in human plasma has beendeveloped prior to this study to establish assay sensitivity,specificity, linearity, and reproducibility.

Pharmacodynamic Endpoints

The PD endpoints, including biomarker assessments, are evaluated duringthe study as follows:

-   -   Blood for potential biomarkers including, but not limited to,        soluble AXL, AXL expression and phosphorylation, GAS6, and        mesenchymal transcription factors Analysis of the sample is        limited to evaluations that are relative to the activity of the        Study Drug or biomarkers of underlying disease.        Adverse Effects and Dose Modification

An adverse event (AE) is defined as any untoward medical occurrenceassociated with the use of a drug in humans, whether or not considereddrug related. An AE can therefore be any unfavorable and unintended sign(including an abnormal laboratory finding), symptom, or diseasetemporally associated with the use of a drug, whether or not related tothe drug product.

A suspected adverse reaction is any AE that had a reasonable possibilityof being caused by the drug. Reasonable possibility means there isevidence to suggest a causal relationship between the drug and the AE.

An unexpected AE or unexpected suspected adverse reaction is an AE orsuspected adverse reaction not known to those skilled in the art, notlisted at the specificity or severity that has been observed, or notconsistent with the risk information described in the protocol.

Toxicities are assessed according to the NCI CTCAE, v5.0. When the NCICTCAE grade is not available, the following toxicity grading: mild,moderate, severe, life threatening, or fatal, may be used.

TABLE 25 Grades of Toxicities GRADE 1 - Mild: Asymptomatic or mildsymptoms; clinical or diagnostic observations only; intervention notindicated GRADE 2 - Moderate: Minimal, local or noninvasive interventionindicated; limiting age-appropriate instrumental ADL^(a) GRADE 3 -Severe: Medically significant but not immediately life-threatening;hospitalization or prolongation of hospitalization indicated; disabling;limiting self-care ADL^(b) GRADE 4 - Life Life-threatening consequences;urgent Threatening: intervention indicated GRADE 5 - Fatal Death relatedto AE ^(a)Instrumental activities of daily living (ADLs) refer topreparing meals, shopping for groceries or clothes, using the telephone,managing money, etc. ^(b)Self-care ADLs refer to bathing, dressing andundressing, feeding self, using the toilet, taking medications, and notbedridden.

Relationship of the AE to the study drug should be defined as follows:

Unrelated: AE is clearly not related to the study drug (ie, there is notemporal association and no other possible cause [intercurrent illness,medication]) Unlikely: AE is doubtfully related to the study drugPossibly: AE may be related to the study drug Probably: AE is likelyrelated to the study drug Definitely: AE is clearly related to the studydrug

A serious adverse event (SAE) is defined as any suspected adversereaction at any dose that suggests a significant hazard,contraindication, side effect, or precaution, and results in thefollowing outcomes:

-   -   Death;    -   A life-threatening event (ie, the patient is at immediate risk        of death from the event as it occurred);    -   An event that is persistently, significantly, severely, or        permanently disabling, or requires intervention to prevent such        disability;    -   An event that requires inpatient hospitalization or prolongs        hospitalization;    -   A congenital abnormality/birth defect; or    -   A medically significant event that may jeopardize the patient or        may require intervention to prevent 1 of the other outcomes        listed above.

Example 50: Route for the Synthesis of M3 (SULF-1) and M4 (SULF-2)

Metabolites M3 and M4 were prepared according to the following schemes.

Example 51: Solvent and Slurry Screening of Crystalline Forms

The experimental conditions and the brief results acquired from thesolvent screening and the slurry screening are listed in Tables 3-6. Thevariable organic solvents and those mixtures with water were used assolvent systems. From these screening studies, ten crystal forms, FormA′, A, B, C, D, E, F, G, H and I, were assigned by XRPD patterns asshown in FIG. 98. The thermal behavior (DSC/TGA charts) for thesecrystals are also shown in FIGS. 99A-99I. The moisture sorptionisotherms for Form A′, A, B, C, and D are provided in FIG. 116.

Form A

The solvent screen was conducted as shown in Table 4. Form A was nottransformed and was recrystallized in almost the organic solvent systemswithout H₂O. Two types of the stoichiometric crystals, which wereindicated from initial studies, were respectively used in the slurryscreen as shown in Tables 3 and 5. They were maintained in almost thesolvent systems except for H2O during the screen. This indicated thatForm A was basically stable and dominant form. Form A was transformed toForm B in H₂O, like as that observed in the solvent screen. The thermalanalysis charts for the two types of Form A were shown in FIG. 99A. Thesignificant weight-loss were observed for these batches with the highermelting point than that expected. The moisture sorption isotherm showedthat these Form A were hygroscopic as shown in FIG. 116, and theadditional XRPD analysis as shown in FIG. 117 found that Form A wasmaintained after the moisture absorption/desorption cycle. Nosignificant changes were observed during the stability studies as shownin Table 26.

TABLE 26 Summary of Stability Results Form Initial 40° C. 40° C./75% RH60° C. 60° C./75% RH A′ 97.9% 98.1% 98.0% 97.8% 98.1% A 99.3% 99.3%99.3% 99.3% 99.3% D 98.4% 98.2% 98.4% 98.2% 98.2%

The two types of the stoichiometric crystals of Form A were finallyidentified since the XRPD pattern of the 2 batches of compound ofstructure (I) tartrate salt were same but the quantitative analyses byion chromatography were different. The ratios of free base and tartaricacid for these two types of stoichiometric crystal were 1:1.5 (Form A′)and 1:2 (Form A). The Raman spectra of Form A were consistent as shownin FIG. 118.

Form B

In the solvent and slurry screen, Form A was transformed to Form B bybeing recrystallized/suspended in H₂O, as shown in Table 4 and Table 5.The XRPD pattern of Form B was similar to that of Form A but it seemedto be contaminated with another crystal form as shown in FIG. 98. About4%-weight loss and a broad endotherm peak derived from adheredwater/solvent was observed and the melting point was over 130° C. asshown in FIG. 99B. The moisture sorption isotherm showed that Form B washygroscopic as shown in FIG. 116. The quantitative analysis by ionchromatography indicated that the stoichiometry of freebase and tartaricacid was 1:1.2. The representative Raman spectrum was shown in FIG. 118,but the Raman spectra varied depending on the measured area of thematerial. This result strongly suggested that Form B was mixture of FormA and another form.

Form C

In the solvent screen, as shown in Table 4, Form C was formed by therecrystallization of Form A with the mixture of H₂O and alcohol, such asmethanol and 2-propanol. About 10%-weight loss and a broad endothermpeak was observed in the thermal analysis chart as shown in FIG. 99C.That suggested that Form C might be a solvate with alcohol and thealcohol was eliminated depending on the increase of temperature. Themoisture sorption isotherm as shown in FIG. 116 indicated that Form Cwas hygroscopic.

Form D

Form D was not formed in the solvent screen from Form A. Form D used inthe slurry screen was not transformed in ethanol and 2-propanol, butdone to various forms, E, F, G, H and I, in the other solvents, as shownin Table 6. This data indicated that Form D would be difficult tocontrol in the manufacturing process. About 3%-weight loss and a broadendotherm peak due to adhered water/solvent was observed in the thermalanalysis chart as shown in FIG. 99D. The moisture sorption isotherm asshown in FIG. 116 indicated that Form D was hygroscopic. The additionalXRPD analysis as shown in FIG. 117 found that Form D was transformed tothe other forms after the moisture absorption/desorption cycle. Nosignificant changes were observed during the stability studies as shownin Table 26. The stoichiometry of freebase and tartaric acid is 1:1.

Form E

Form E was formed in the slurry screen from Form D only in methanol atroom temperature, as shown in Table 6. About 4%-weight loss was observedin the thermal analysis chart as shown in FIG. 99E.

Form F

Form F was formed in the slurry screen from Form D in the mixture ofalcohol and H₂O at room temperature and 50° C. as shown in Table 6.About 6%-weight loss was observed in the thermal analysis chart as shownin FIG. 99F.

Form G

Form G was formed in the slurry screen from Form D in H₂O at roomtemperature and 50° C. as shown in Table 6. The thermal analysis chartwas provided in FIG. 99G. No further studies could be done because ofthe insufficient amount of sample.

Form H

Form H was formed in the slurry screen from Form D only in Methanol-H₂O(5:1) at room temperature, as shown in Table 6. The thermal analysischart is provided in FIG. 99H.

Form I

Form I was formed in the slurry screen from Form D only inacetnirnile-H₂O (10:1) at 50° C., as shown in Table 6. Thermal analysisdata is provided in FIG. 99I.

CONCLUSION

The polymorph screen revealed that the compound of structure (I)tartrate salt had the nine crystal forms involving solvates. Among them,Form A could be the most suitable form for the tartrate sale when itsstoichiometry is well-controlled in the manufacturing process, in termsof the dominance in the screens and the acceptable solid-formproperties.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification or theattached Application Data Sheet are incorporated herein by reference, intheir entirety to the extent not inconsistent with the presentdescription.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

The invention claimed is:
 1. A method of treating a cancer, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a crystalline form of a tartrate salt of thecompound of structure (I):

wherein the cancer is: a solid tumor selected from non-small cell lungcancer (NSCLC), colorectal carcinoma (CRC), ovarian cancer, melanoma,breast carcinoma, neuroendocrine carcinoma, prostate adenocarcinoma,cholangiocarcinoma, uterine carcinoma, pancreatic cancer, lungadenocarcinoma, lung squamous cell carcinoma, urethral cancer,urothelial cancer, ductal cancer, endometrial cancer or renal carcinoma;or a hematological cancer selected from multiple myeloma,myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), acutelymphoblastic leukemia (ALL), acute lymphocytic leukemia, chroniclymphogenous leukemia, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), mantle cell lymphoma, diffuse large B-celllymphoma, follicular lymphoma, or non-Hodgkin's lymphoma.
 2. The methodof claim 1, wherein the crystalline form is a crystalline form of atartrate salt having a molar ratio of tartaric acid to the compound ofstructure (I) of about 2:1.
 3. The method of claim 2, wherein thecrystalline form comprises crystalline Form A.
 4. The method of claim 1,wherein the crystalline form of a tartrate salt is administered to thesubject in a composition comprising the crystalline form and apharmaceutically-acceptable carrier or diluent.
 5. The method of claim4, wherein the composition is formulated for oral administration.
 6. Themethod of claim 1, comprising administering an oral daily dose of thecrystalline form.
 7. The method of claim 6, wherein the oral daily doseof the crystalline form of a tartrate salt is selected from: i) about 20mg to about 100 mg of the crystalline form of a tartrate salt; ii) about25 mg to about 75 mg of the crystalline form of a tartrate salt; iii)about 25 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about58 mg, about 75 mg, or about 100 mg of the crystalline form of atartrate salt; or iv) about 1.0, 1.5, 3.0, 6.0, 9.0, 12.0, 16.0, 21.0,28.0, 37.0, 49.0, or 65.0 mg/m² of the crystalline form of a tartratesalt.
 8. The method of claim 6, wherein the oral daily dose isadministered once daily (qd).
 9. The method of claim 6, wherein the oraldaily dose is a flat dose.
 10. The method of claim 1, wherein the methodcomprises one or more treatment cycles.
 11. The method of claim 10,wherein each treatment cycle consists of about 28 days, optionally,wherein in each treatment cycle, the crystalline form of a tartrate saltis administered to the subject: i) continuously for 28 days, or ii)daily for 21 days, followed by a drug holiday period of seven days. 12.The method of claim 1, further comprising administering an effectiveamount of one or more other therapeutic agents to the subject.
 13. Themethod of claim 12, wherein the one or more other therapeutic agents areselected from one or more of: (i) an immunotherapy agent; (ii) an immunecheckpoint inhibitor; (iii) a tyrosine kinase inhibitor (TKI); (iv) aBTK inhibitor; (v) a CDK inhibitor; (vi) a CDK-9 inhibitor; (vii) one ormore of a PD-1 inhibitor, a PDL-1 inhibitor and a CTLA-4 inhibitor;(viii) a pyrimidine analog; (ix) a BCL-2 inhibitor; (x) an EGFRinhibitor; (xi) one or more of pembrolizumab and nivolumab; (xii) one ormore of atezolizumab, avelumab, and durvalumab; (xiii) a platinum-basedchemotherapeutic agent; (xiv) one or more of carboplatin, gemcitabine,bevacizumab, topotecan, rucaparib, olaparib, niraparib, nivolumab,pembrolizumab, atezolizumab, avelumab, durvalumab, and ipilimumab; (xv)an antimetabolite; and (xvi) venetoclax.
 14. The method of claim 12,wherein the one or more other therapeutic agents include a tyrosinekinase inhibitor (TKI), optionally, wherein the TKI is an EGFR TKI. 15.The method of claim 12, wherein the one or more other therapeutic agentsinclude an anti-PD-1 agent, optionally, wherein the anti-PD-1 agent ispembrolizumab.
 16. The method of claim 12, wherein the one or more othertherapeutic agents include a pyrimidine analog, optionally, wherein thepyrimidine analog is ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, or floxuridine.17. The method of claim 12, wherein the one or more other therapeuticagents include an anti-metabolite.
 18. The method of claim 12, whereinthe one or more other therapeutic agents include a BCL-2 inhibitor,optionally, wherein the BCL-2 inhibitor is venetoclax.
 19. The method ofclaim 1, wherein the cancer is a solid tumor selected from the groupconsisting of non-small cell lung cancer (NSCLC), colorectal carcinoma(CRC), ovarian cancer, melanoma, breast carcinoma, neuroendocrinecarcinoma, prostate adenocarcinoma, cholangiocarcinoma, uterinecarcinoma, pancreatic cancer, lung adenocarcinoma, lung squamous cellcarcinoma, urethral cancer, urothelial cancer, ductal cancer,endometrial cancer and renal carcinoma.
 20. The method of claim 19,wherein the solid tumor is EGFR⁺ NSCLC, BRAF-mutated colorectalcarcinoma (CRC), KRAS-mutated CRC, NRAS-mutated CRC, persistent orrecurrent ovarian cancer, platinum-refractory or platinum-resistantovarian cancer, clear cell ovarian cancer, BRAF-wildtype melanoma, orBRAF-mutated melanoma.
 21. The method of claim 19, wherein the solidtumor is an advanced solid tumor, or is a solid tumor which isclassified as a resistant, recurrent, refractory, or relapsed solidtumor.
 22. The method of claim 19, wherein the solid tumor has showndisease progress after receiving one or more prior cancer therapy. 23.The method of claim 22, wherein the one or more prior cancer therapycomprises a hormone therapeutic agent, a chemotherapeutic agent, animmunotherapeutic agent, or a cell surface receptor inhibitor.
 24. Themethod of claim 23, wherein the one or more prior cancer therapycomprises: i) a tyrosine kinase inhibitor (TKI), optionally, wherein theTKI is an EGFR TKI; or ii) one or more prior cancer therapy comprises aplatinum-containing agent; or wherein the solid tumor is resistant to atleast one of: an immunotherapy, a BCL-2 inhibitor therapy, or TKItherapy, and wherein the method further comprises subjecting the subjectadditionally to the same immunotherapy, BCL-2 inhibitor therapy, or TKItherapy, respectively, optionally, wherein the solid tumor is resistantto an immunotherapy, and wherein the method further comprises subjectingthe subject additionally to the same immunotherapy, optionally, whereinthe immunotherapy comprises one or more of a PD-1 inhibitor, a PD-L1inhibitor, or a CTLA4 inhibitor.
 25. The method of claim 19, furthercomprising administering an effective amount of one or more othertherapeutic agents to the subject, optionally, wherein the one or moreother therapeutic agents include: a i) tyrosine kinase inhibitor (TKI),optionally, wherein the TKI is an EGFR TKI; and ii) an anti-PD-1 agent,optionally, wherein the anti-PD-1 agent is pembrolizumab.
 26. The methodof claim 19, comprising administering an oral daily dose of thecrystalline form of a tartrate salt selected from: i) about 20 mg toabout 100 mg of the crystalline form of a tartrate salt; ii) about 25 mgto about 75 mg of the crystalline form of a tartrate salt; or iii) about25 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 58 mg,about 75 mg, or about 100 mg of the crystalline form of a tartrate salt.27. The method of claim 1, wherein the cancer is a hematological cancerselected from multiple myeloma, myelodysplastic syndrome (MDS), acutemyelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acutelymphocytic leukemia, chronic lymphogenous leukemia, chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma,diffuse large B-cell lymphoma, follicular lymphoma, or non-Hodgkin'slymphoma.
 28. The method of claim 27, wherein the hematological canceris acute myelogenous leukemia (AML).
 29. The method of claim 27, whereinthe hematological cancer is associated with a TP53 mutation.
 30. Themethod of claim 27, comprising administering an oral daily dose of thecrystalline form of a tartrate salt selected from: i) about 20 mg toabout 100 mg of the crystalline form of a tartrate salt; ii) about 25 mgto about 75 mg of the crystalline form of a tartrate salt; and iii)about 25 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about58 mg, about 75 mg, or about 100 mg of the crystalline form of atartrate salt.
 31. The method of claim 27, further comprisingadministering to the subject one or more other therapeutic agents,wherein the one or more other therapeutic agents includes one or morechemotherapeutic agents.
 32. The method of claim 31, wherein the one ormore chemotherapeutic agents includes a pyrimidine analog.
 33. Themethod of claim 32, wherein the pyrimidine analog is ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, or floxuridine.
 34. The method of claim 31,wherein the one or more chemotherapeutic agents includes ananti-metabolite.
 35. The method of claim 31, wherein the one or morechemotherapeutic agents includes a BCL-2 inhibitor, optionally whereinthe BCL-2 inhibitor is venetoclax.
 36. The method of claim 1, whereinthe subject is a human.